System for processing cells and container for use therewith

ABSTRACT

A system for processing cells is disclosed. The system may include a container having an inner wall, a plunger adapted to be slidably positioned within the container, at least one inlet port through which a fluid can enter the container, at least one outlet port through which a fluid can exit the container, at least one effluent port through which an effluent can exit the container, a first check valve in fluid connection with the inlet port and a second check valve in fluid connection with the effluent port. The plunger may include a filter that allows fluid to pass therethrough but prevents cells from passing therethrough. Rearward motion of the plunger may be adapted to draw fluid into the system via the inlet port and forward motion of the plunger may be adapted to force effluent out of the system via the effluent port.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 11/460,635,filed Jul. 28, 2006, now U.S. Pat. No. 7,713,232, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/771,206,filed Feb. 7, 2006, U.S. Provisional Patent Application Ser. No.60/742,224, filed Dec. 5, 2005, and U.S. Provisional Patent ApplicationSer. No. 60/734,035, filed Nov. 4, 2005, the disclosures of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the delivery of agents such astherapeutic agents to tissue and, particularly, to the delivery of cellsto tissue.

The following information is provided to assist the reader to understandthe invention disclosed below and the environment in which it willtypically be used. The terms used herein are not intended to be limitedto any particular narrow interpretation unless clearly stated otherwisein this document. References set forth herein may facilitateunderstanding of the present invention or the background of the presentinvention. The disclosures of all references cited herein areincorporated by reference.

The treatment of disease by the injection of living cells into a body isexpanding rapidly. There are many types of cells being used to treat anequally diverse set of diseases, and both types of cells and diseaseconditions are expanding rapidly. Xenogeneic cell therapies involveimplantation of cells from one species into another. Allogeneic celltherapies involve implantation from one individual of a species intoanother individual of the same species. Autologous cell therapiesinvolve implantation of cells from one individual into the sameindividual.

In an example of an allogeneic cell therapy, current phase II clinicaltrials of SPHERAMINE® by Titan Pharmaceutical of San Francisco, Calif.and Schering AG of Berlin, Germany, retinal pigment epithelial cells areharvested from eyes in eye banks, multiplied many fold in culture mediumand placed on 100 micrometer diameter gelatin spheres. The sphericalmicroscopic carriers or microcarriers greatly enhance the cells'survival when injected in the brain. The carriers are injected throughneedles into the putamen in the brain. The animal precursor work isdescribed in several patents, including U.S. Pat. Nos. 6,060,048,5,750,103, and 5,618,531, the disclosures of which are incorporatedherein by reference. These patents describe many types of cells,carriers, and diseases that can be treated via the disclosed methods. Ina rat, about 20 microliters (ul) of injected cells on carriers issufficient to restore dopamine production to a damaged rat brain. Thetherapy was injected at the rate of 4 ul/min. This dosage scales to atotal injected volume of 0.5 ml in the human brain, although it willhave to be distributed over a larger region, probably via multipleindividual injections on the order of the 20 ul mentioned above. Celltherapies for the brain and nervous system are discuss further below.

An example of an autologous cell therapy involves the harvesting ofmesenchymal stem cell from a patient's bone marrow, concentration of thestem cells, and injection of the cells and other blood components intothe heart muscle during open-heart surgery. Further examples includecatheter delivered cell therapies, especially to the heart, laparoscopicdelivered therapies, and transcutaneous therapies

In external cell therapy for the heart, volumes of about 0.5 to 1.0 mlare injected into a beating heart. A multi-milliliter syringe is used tohold and deliver the injectate under manual activation. A challenge ispresented in that when the heart is contracting, during systole, thetissue becomes relatively hard and tense. In diastole, the tissuerelaxes. It is very difficult for a human to time and control a handinjection so that the proper volume is injected all in one period ofdiastole. In practice, an indeterminate amount of the injectate cansquirt or leak out the needle track and is presumably wasted. Inaddition, it is desirable to uniformly and thoroughly treat the targetareas of the heart, and to avoid puncturing the major blood vesselstraversing the outside of the heart. These results can also be difficultto achieve with current manual injection practices. With the currentstate of practice, scar tissue is not injected or treated because itdoes not respond well, and the growth that does occur can sometimescreate dangerous electrical conduction abnormalities.

Cell therapies are generally delivered by hand injection through aneedle or catheter. The benefits of hand or manual injection areconceptual simplicity and familiarity for the doctor. However thesimplicity is misleading. Many of the parameters of the injection arenot and cannot be controlled or even repeated by that doctor, let aloneby other doctors. Flow rate is, for example, very difficult to controlmanually, especially at low flow rates. The stick slip friction ofnormal syringes exacerbates this problem. Volume accuracy depends uponmanual reading of gradations, which is physically difficult whilesqueezing the syringe and susceptible to human perceptual ormathematical errors. The use of common infusion pumps limits delivery togenerally slow and very simple fluid deliveries. Infusion pumps have noability to provide automatic response or action to the injection basedupon any physiological or other measurement or feedback.

Tremendous variations in manually controlled injectate delivery canproduce proportionally wide variations in patient outcomes. In clinicaltrials, this variation is undesirable because it increases the number ofpatients and thus cost and time needed to establish efficacy. In longterm therapeutic use, such variation remains undesirable as some peoplecan receive suboptimal treatment.

FIG. 1A illustrates the current manual state of the art. Cells are takenfrom a bag or other storage or intermediate container and loaded into asyringe. This procedure involves making and breaking fluid connectionsin the room air which can compromise sterility, or requires a specialbiological enclosure to provide class 100 air for handling. The syringeis then connected to a patient interface or applicator, which iscommonly a needle, catheter, or tubing that is then connected to aneedle or catheter. For many procedures, there is some type of imagingequipment involved in guiding the applicator or effector to the correctpart of the body. For example, the imaging equipment can include X-rayfluoroscopy, CT, MR, ultrasound, or an endoscope. The physician viewsthe image and places the applicator by hand. In some neurologicalprocedures, a stereotaxic (or stereotactic) positioner or head frame isused to guide the applicator to the target tissue, deep in the brain,based on coordinates provided by the imaging system. The patientphysiological condition is often monitored for safety, especially whenthe patient is under general anesthesia.

As discussed briefly above, medical research has demonstrated utility ofimplantation of cells into the brain and central nervous system astreatment for neurodegenerative disorders such as Parkinsons,Alzheimers, stroke, motor neuron dysfunction experienced, for example,by victims of spinal cord injury. As with other cell therapies, themechanisms of repair are not well understood, but the injection of cellsinto damaged parenchymal tissue has been shown to recruit the body'snatural repair processes and to regenerate new functional tissue as wellas the cells themselves living and integrating into the tissue.

As with other cell delivery techniques described above, a longrecognized, but unmet need in this field is a set of methods and devicesthat can provide precise, repeatable and reliable control of dosage ofthese therapeutic agents in actual clinical settings. Current manualapproaches (as summarized above and in connection with FIG. 1A) do notaddress all of the needs required by new procedures. For example, thereare no good methods for ensuring the parameters of cell viability,including spatial distribution, cell quantity, metabolic and electricalactivity, in real time during the entire implantation procedure. Thesevariables are affected by cell storage conditions, by the fluid dynamicsof an injection (for example, flow, shear stresses or forces, fluiddensity, viscosity, osmolarity, gas concentration), by thebiocompatibility of materials, and by the characteristics of surroundingtissues and fluids.

Deleterious effects of flow of cells through fluid paths are also notwell addressed in current techniques. For example, Luer standardconnectors are used almost universally in the current medical practice,including in fluid paths for cell delivery. An example of a lurestandard connector 1 is show in FIG. 1B. FIG. 1B is taken from thestandard ISO 594-1-1986, figure number 2. As the tapered sections of themale 1 a and female 1 b connectors mate, a dead space is created asindicated by 1 c. In addition, the sharp transition in the fluid path atthe end of the male luer, as indicated at 1 d, can create turbulence andincrease shear stress in the fluid and on the cells, resulting in celldamage or even death. Moreover, similar problems exist in commonly usedfluid path elements other than connectors.

There are current methods for delivery of chemotherapeutic agentsdirectly to the brain and other central nervous system structures (CNS)including, for example, convection enhanced delivery (CED) and otherdirect injection by needles, catheters, and syringes into CNSstructures. These direct injections are an alternative to less effectiveintravenous drug delivery methods. Other approaches to drug delivery inthe CNS include the placement of drug-impregnated hydrogel wafers(Gliadel®) directly into brain tissue for extended periods of time aftertumor excision. In the case of Parkinson's disease treatment,dopamine-producing cells are assembled onto gelatin beads (SPHERAMINE®,Titan Pharmaceuticals), which are hand-injected through precisionsyringes into the brain. The effectiveness of these methods is typicallymonitored long after initial treatment with non-invasive imaging (CT,MR).

Examples of systems and methods for convection enhanced delivery to thebrain and other solid tissue structures is described in U.S. Pat. No.5,720,720, the disclosure of which is incorporated herein by reference.Although the '720 patent discloses methods of injecting liquidmedications based on a biomechanical model of tissue, it does notaddress problems unique to the delivery of complex slurries of fragileneural cells. U.S. Pat. No. 6,599,274, the disclosure of which isincorporate herein by reference, discloses methods of cell delivery tothe brain using catheter injection systems. Control systems aredescribed in which the distribution and function of therapeutic cells,growth factors, or other proteins are monitored by various techniques ofimaging, physical, chemical, and electrical measurement. The '274 patentmentions closed loop, real-time control of the cell infusion processbased on imaging and measured properties. However, the '274 patent doesnot describe how the elements of a controlled cell storage system worktogether with an injection system to guarantee delivery of viable cellsof correct dosage and associated growth factors into tissues of the CNS.U.S. Pat. No. 6,758,828 describes a cell storage system for maintainingthe viability of cells injected into tissue, but does not describe anintegrated control system for monitoring the viability of cells as theyenter the patient and take up residence in the parenchyma, nor does itdescribe how cell viability can be monitored in vivo.

U.S. Pat. No. 6,749,833 discloses methods to sustain the viability ofcells by limiting damage resulting from shear stresses during fluidflow. An apparatus is described which allows for continuous bolus flowor peristaltic flow by reducing these shear forces. It is not clear fromthe '833 patent how the viability of cells is to be measured afterdelivery of the cells into living tissue. U.S. Pat. Nos. 6,572,579,6,549,803 and 6,464,662 attempt to address the problem of distributing adose of biologically active material into tissue by means of directcatheter injection.

In addition to application of cell therapies to internal tissues such aheart tissue, brain tissue and central nervous system tissue, celltherapies have also recently been applied to skin. Dermatologists havebeen injecting drugs into the skin for years. Recently, injections ofcollagen, which can be thought of as a cell-less tissue, have becomepopular. Moreover, Intercytex of Cambridge UK has developed the abilityto inject autologous dermal papilla cells for the growth of hair totreat baldness. The cells are harvested from a person, multiplied inculture, and then reimplanted into the same person. The implantationrequires about 1000 injections of 1 microliter each.

Various aspect of delivery of agent to tissue and related aspects arealso discussed in U.S. Pat. Nos. and Patent Application Nos. 5,720,720,5,797,870, 5,827,216, 5,846,225, 5,997,509, 6,224,566, 6,231,568,6,319,230, 6,322,536, 6,387,369, 6,416,510, 6,464,662, 6,549,803,6,572,579, 6,599,274, 6,591,129, 6,595,979, 6,602,241, 6,605,061,6,613,026, 6,749,833, 6,758,828, 6,796,957, 6,835,193, 6,855,132,2002/0010428, 2002/0082546, 2002/0095124, 2003/0028172, 2003/0109849,2003/0109899, 2003/0225370, 2004/0191225, 2004/0210188, 2004/0213756,and 2005/0124975, as well as in, PCT Published International PatentApplication WO2000/067647, EP1444003, the disclosures of which areincorporated herein by reference.

Although various devices, systems and methods have been developed fordelivery of agents, including therapeutic agent, to various types oftissue, it remains desirable to develop improved devices, systems andmethods for delivering agents to tissue and, particularly, fordelivering therapeutic cells to tissue.

The present invention, along with the attributes and attendantadvantages thereof, will best be appreciated and understood in view ofthe following detailed description taken in conjunction with theaccompanying drawings.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a system for injecting aninjectate into patient, including: a first pressurizable container forholding the injectate; a patient interface in fluid connection with thefirst pressurizable container, the patient interface being adapted topass the injectate into tissue of the patient; a powered injector inoperative connection with the first pressurizable container topressurize the injectate; a controller system in operative connectionwith powered injector; and a stereotactic localization frame adapted tobe placed in operative connection with the patient interface to assistin controlling localization of the patient interface.

The system can further include a communication system in connection withthe controller system. The communication can, for example, provideinformation to the controller system of at least one measured parameter.The controller system is preferably adapted to transmit a control signalbased at least in part on the measure parameter to control the poweredinjector. The system can also include at least one monitoring systemadapted to measure at least one physiological property of the patient.The communication system can be in connection with the monitoring systemto provide information of the measured value of the at least onephysiological property to the controller system. The controller systemcan, for example, be adapted to transmit a control signal based at leastin part on the measured value of the least one physiological property.

The system can also include an imaging system adapted to image a regionof the patient to which the injectate is delivered. The communicationsystem can, for example, be in connection with the imaging system toprovide information of a measured property from the imaging system tothe controller. In this embodiment, the controller can be adapted totransmit a control signal based at least in part on the measuredproperty from the imaging system.

The system can also at least one sensor to measure at least one propertyof the injectate. The sensor being can be connection with thecommunication system to provide information of the measured injectionfluid property to the controller. The at least one property of theinjectate can, for example, provides a measurement of shear forces onthe injectate. The at least one property of the injectate can, forexample, beat least one of flow rate or pressure.

In several embodiments, the first pressurizable container is a firstsyringe having a first plunger slidably disposed therein to pressurizethe injectate. The injector is adapted to effect movement of the firstplunger.

The powered injector can, for example, be in operative connection with asecond pressurizable container. The second pressurizable container canbe in fluid connection with the first pressurizable container such thatpressurized fluid from the second pressurizable container is operable topressurize the injectate within the first pressurizable container. Inseveral embodiments, the first pressurizable container is a firstsyringe having a first plunger slidably disposed therein to pressurizethe injectate, and pressurized fluid from second pressurizable containeris operable to effect movement of the first plunger. The secondpressurizable container can be a second syringe having a second plungerslidably disposed therein.

The system can also include a sterile containment system adapted toencompass at least a portion of the first pressurizable container and aportion of the powered injector.

In another aspect, the present invention provides a container adapted tostore and transport an injectate. The container is adapted to have afluid introduced therein and effluent removed therefrom to effectprocessing of the injectate. The container can, for example, be adaptedto be subjected to freezing and thawing.

The container can include a first port at a first axial position withinthe container and at least a second port at a second axial positionwithin the container, wherein the first axial position and the secondaxial position are different. The first port and the second port can,for example, be formed monolithically with the container. The first portcan also be on a distal end of a first tube extending through an endclosure of the container, and the second port can be on the distal endof a second tube extending through the end closure the container. In oneembodiment, the end closure is a septum, the first tube is a firstpiercing member and the second tube is a second piercing member. Thecontainer can, for example, encompass viable cells within a first fluid.At least one of the first axial position and the second axial positioncan be above an axial position of cells settled to a bottom of thecontainer. The cells can, for example, be retinal pigment epithelialcells supported on microspheres, mesenchymal stem cells, multipotentadult progenitor cells, embryonic stem cells, cardiac precursor cells,cardiac cells, beta-islet precursor cells, beta-islet cells, neuralprecursor cells, or neural cells.

In another embodiment, the container includes a divider within thecontainer to create a first fluid path on a first side of the dividervia which fluid can enter the container and a second fluid path on asecond side of the divider via which fluid can exit the container. Thesecond side fluid path includes at least one filter through which fluidcan pass but through which at least one component of the injectatecannot pass. As discussed above, the container can encompasses viablecells within a first fluid. The filter preferably prevents cells frompassing therethrough. Once again, the cells can, for example, be retinalpigment epithelial cells supported on microspheres, mesenchymal stemcells, multipotent adult progenitor cells, embryonic stem cells, cardiacprecursor cells, cardiac cells, beta-islet precursor cells, beta-isletcells, neural precursor cells, or neural cells.

In a further aspect, the present invention provides a method ofprocessing cells prior to delivery thereof including the step ofcontacting the cells with at least one fluid to decrease theconcentration of a hibernation solution in which the cells aretransported wherein the occurrences of exposure of the cells tonon-sterile air is minimized and the duration of any occurrence ofexposure to non-sterile air is minimized. The cells can be transportedin container adapted to store and transport the cells. The container ispreferably adapted to have the at least one fluid introduced therein andeffluent removed therefrom. The at least one fluid can include a buffersolution. A closed system can, for example, be used in the processing.The closed system can, for example, include a source of the first fluidadapted to be placed in fluid connection the container. As discussedabove, the container can include a first port for introduction of the atleast one fluid and a second port for removal of effluent.

The closed system can include a pump system to effect flow of the atleast on fluid into the container and effluent out of the container. Theclosed system can further include a first one-way valve in fluidconnection with the first port and a second one-way valve in fluidconnection with the second port.

The container can further include a third port adapted to provide airinto the container and a sterile filter in fluid connection with thethird port so that air can move in and out as fluid level changes in thecontainer while maintaining sterility.

In several embodiments, the first port has a first tube extendingtherethrough to a first length within the container and the second porthas a second tube extending therethrough to a second length within thecontainer. The first length is greater than the second length, such thatwhen the container is in a generally upright position and cells aresettled to the bottom thereof, the end of the first tube is within thecells and the end of the second tube is above the level of the cells.

In another aspect, the present invention provides a system for use inthe processing of cells encompassed in a container. The system includesat least a first fluid path adapted to introduce at least one fluid intothe container and at least a second fluid path adapted to removeeffluent from the container. The system can further include a pumpsystem to effect flow of the fluid into the container and flow ofeffluent from the container. The system can also include a valve systemto control at least flow of the fluid into the container and flow ofeffluent from the container. The valve system can include at least afirst one way valve in fluid connection with the first fluid path and atleast a second one way valve in fluid connection with the second fluidpath.

The system can also include a controller in operative connection with atleast one of the pump system or the valve system. The controller can,for example, include a computer processor (for example, a microprocessoror a PC).

The system can further include a third fluid path adapted to be placedin fluid connection with the container and be placed in connection witha fluid delivery system in which the cells can be loaded for delivery toa patient. The system can also include at least a fourth fluid pathadapted to be placed in fluid connection with the container to remove asample from the container for analysis. Likewise, the system can alsoinclude at least a fifth fluid path adapted to introduce air into thecontainer. A filter can be placed in fluid connection with the fifthfluid path to assist in maintaining sterility.

The system can further include a source of the fluid in fluid connectionwith the first fluid path.

In another aspect, the present invention provides a system forprocessing cells (and/or other injectate components) comprising acontainer and a plunger adapted to be slidably positioned within thecontainer. The system includes at least one inlet port through which afluid can enter the system and at least one effluent port through whichan effluent can exit the system. The plunger section forms a sealingengagement with the inner wall of the container such that rearwardmotion of the plunger is adapted to draw fluid into the system via theinlet and forward motion of the plunger is adapted to force effluent outof the system via the effluent port.

The effluent port can also be adapted to effect delivery of cellstherethrough to a patient. The system can also include an outlet portadapted to effect delivery of cells therethrough to a patient. A filtercan be placed in fluid connection with the effluent port to preventcells from exiting via the effluent port in such an embodiment.

The system can further include a first check valve in fluid connectionwith the inlet port and a second check valve in fluid connection withthe effluent port.

The plunger can include a filter disposed therein that allows fluid topass therethrough but prevents cells from passing therethrough. Thefilter can, for example, separate the cells from the effluent port. Thefilter can also separate the cells from the inlet port.

In embodiments including an outlet as described above, the outlet portcan adapted to be closed during processing of the cells during whichfluid enters the system via the inlet port and effluent exits the systemvia the effluent port. Moreover, the effluent port can be adapted to beclosed when the outlet port is opened to deliver cells therethrough.

The inlet port can, for example, be in fluid connection with a passagethrough the plunger so that the fluid can enter the plunger and passthrough the filter. The effluent port can likewise be in fluidconnection with a passage through the plunger so that the effluent canpass through the filter and exit the efluent port. The plunger includesa sealing member adapted for form a sealing engagement with an interiorwall of the container.

In still a further aspect, the present invention provides a plunger foruse in connection with a container encompassing cells (and/or otherinjectate component(s)) to effect processing of the cells (and/or otherinjectate component(s)). The plunger includes a filter through whichfluid can pass but cells cannot pass and a sealing member adapted forform a sealing engagement with an interior wall of the container. Theplunger can also include an inlet port to introduce fluid into theplunger to pass through the filter to enter the container and aneffluent port through which effluent can flow through the filter to exitthe container. The plunger can further include a one way valve in fluidconnection with the inlet port. The plunger can likewise include a oneway valve in fluid connection with the effluent port.

The present invention, along with the attributes and attendantadvantages thereof, will best be appreciated and understood in view ofthe following detailed description taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a block diagram of an embodiment of a currentlyavailable system and method for injection of cells.

FIG. 1B illustrates an embodiment of a standard luer connector.

FIG. 2 sets forth several embodiments of systems of the presentinvention for use in delivery of an injectate or injection fluid, andparticularly an injection fluid containing cells, to a brain of apatient

FIG. 3 sets forth several other embodiments of systems of the presentinvention for delivery of an injection fluid, and particularly cells, tothe heart of a patient.

FIG. 4 illustrates a generalized embodiment of a patient interface ofthe present invention.

FIG. 5A illustrates an embodiment of a fluid path of the presentinvention providing for a gradual transition between inner diameters.

FIG. 5B illustrates an embodiment of a fluid path of the presentinvention including a connector providing for a curved, rounded orradiused transitions.

FIG. 5C illustrates the use of an intervening fitting or connector ofthe present invention to create a smooth transition between tubing and aneedle.

FIG. 5D illustrates use of another fitting, transition or connector ofthe present invention to connect a first section and a second section toprovide for smooth internal diameters in the fluid path.

FIG. 5E illustrates a fitting or connector of the present invention thatprovides for relatively smooth transitions and low fluid loss.

FIG. 5F illustrates another fitting or connector of the presentinvention that provides for relatively smooth transitions and low fluidloss.

FIG. 5G illustrates a comparison of the fitting or connector of FIG. 5Fwith a standard luer connector.

FIG. 5H illustrates another fitting or connector of the presentinvention that provides for relatively smooth transitions and low fluidloss.

FIG. 5I illustrates a luer-type fitting or connector of the presentinvention that provides for relatively smooth transitions and low fluidloss.

FIG. 5J illustrates another luer-type fitting or connector of thepresent invention that provides for relatively smooth transitions andlow fluid loss.

FIG. 5K illustrates another luer-type fitting or connector of thepresent invention that provides for relatively smooth transitions andlow fluid loss.

FIG. 5L illustrates another luer-type fitting or connector of thepresent invention that provides for relatively smooth transitions andlow fluid loss.

FIG. 6A illustrates a relatively low capacitance delivery system of thepresent invention including a braided or otherwise stiffened connectortubing.

FIG. 6B illustrates an embodiment of a delivery system of the presentinvention including silicone tubing or other compliant tubing thatallows only a defined amount of pressure to be delivered before ityields, thereby limiting the system pressure.

FIG. 6C illustrates a delivery system of the present invention includinga separate spring loaded vessel to absorb pressure and to provide anindication to the operator that the desired pressure has been exceeded.

FIG. 6D illustrates a syringe including a spring isolated plungerdesigned such that an operatively connected spring or other biasingmember will not compress under a predefined threshold load.

FIG. 6E illustrates a delivery system of the present invention includinga normally closed, a push-button valve to activate a pump system orinjector system and open fluid passage to a needle.

FIG. 6F illustrates the use of a one-way check valve in a needle of thepresent invention in which the needle is placed in tissue.

FIG. 6G illustrates the use needle of FIG. 6F in which the needleoutside of the tissue.

FIG. 6H illustrates a delivery system of the present invention in whichcapacitance is reduced by reducing the total volume of the system.

FIG. 6I illustrates decreasing of capacitance of a fluid path element byincreasing wall thickness.

FIG. 6J illustrates a transparent or hidden line view an embodiment of asyringe of the present invention in which capacitance is substantiallyreduced or eliminated and wherein the syringe plunger is in a forwardposition.

FIG. 6K illustrates a side view of the syringe of FIG. 6J.

FIG. 6L illustrates a cross-sectional view of the syringe of FIG. 6J.

FIG. 6M illustrates another transparent or hidden line view of thesyringe of FIG. 6J wherein the syringe plunger is in a rearwardposition.

FIG. 6N illustrates a rear view of the syringe of FIG. 6J.

FIG. 6O illustrates an embodiment of an injector or delivery system ofthe present invention.

FIG. 6P illustrates a pressure profile of the system of FIG. 6O withsystem capacitance wherein the patient interface is positioned withintissue.

FIG. 6Q illustrates a pressure profile of the system of FIG. 6O withsystem capacitance wherein the patient interface is removed from tissue.

FIG. 6R illustrates an embodiment of a delivery system or applicator ofthe present invention in which a generally solid component, element orplug is placed within tissue.

FIG. 7A illustrates a schematic diagram of three fluid paths inoperative connection as concentric cylinders.

FIG. 7B illustrates a simple fluid path including one fluid path elementand one fluid path.

FIG. 7C illustrates a fluid path including two fluid path elements.

FIG. 7D illustrates another fluid path including two fluid pathelements.

FIG. 7E illustrates a fluid path in which injectate is pulled back afirst fluid path while a purging or physiological solution is injectedat the same flow rate down a second fluid path.

FIG. 7F illustrates the fluid path elements of FIG. 7A, with anexemplary fluid flow indicated.

FIG. 8A illustrates an embodiment of an injection system of the presentinvention in which a disposable container or syringe can be snappedsecurely and reliably into place with an injector in a simple, two-stepoperation.

FIG. 8B illustrates a cutaway view of the injector system of FIG. 8Ashowing the motor and battery power supply.

FIG. 8C illustrates another embodiment of an injector system of thepresent invention.

FIG. 8D illustrates an embodiment of an injection or fluid deliverysystem of the present invention for use, for example, in connection witha stereotactic localization frame.

FIG. 8E illustrates another embodiment of an injection or fluid deliverysystem of the present invention for use, for example, in connection witha stereotactic localization frame.

FIG. 9A illustrates an embodiment of a handheld switch or controlassembly for use with an injector system.

FIG. 9B illustrates the switch or control assembly of FIG. 9A inoperative connection with the injector system of FIG. 8A.

FIG. 9C illustrates the injector system of FIG. 8A as worn on the arm ofan operator, wherein a sterile barrier surrounds the injector system.

FIG. 9D illustrates the injector system of FIG. 8A adapted to be worn onthe body of an operator.

FIG. 9E illustrates the injector system of FIG. 8A including embodimentof control and display panels.

FIG. 9F illustrates an embodiment of a head mounted display for use inconnection with the injection systems of the present invention.

FIG. 10A illustrates an embodiment of a transport and/or storagecontainer of vial of the present invention adapted to effect cellwashing and/or buffer replacement.

FIG. 10B illustrates another embodiment of a transport and/or storagecontainer of vial of the present invention adapted to effect cellwashing and/or buffer replacement.

FIG. 10C illustrates another embodiment of a transport and/or storagecontainer of vial of the present invention adapted to effect cellwashing and/or buffer replacement.

FIG. 10D illustrates the vial of FIG. 10C with a rounded bottom portion.

FIG. 10E illustrates an embodiment of a closed system of the presentinvention suitable to effect cell washing and/or buffer replacement.

FIG. 10F illustrates another embodiment of a transport and/or storagecontainer of vial of the present invention adapted to effect cellwashing and/or buffer replacement and including a pierceable septum.

FIG. 11A illustrates an embodiment of a devices of the present inventionadapted to effect cell washing and/or buffer replacement and cellinjection.

FIG. 11B illustrates another embodiment of a devices of the presentinvention adapted to effect cell washing and/or buffer replacement andcell injection.

FIG. 11C illustrates another embodiment of a device of the presentinvention adapted to effect cell washing and/or buffer replacement andcell injection wherein buffer is being drawn into the device.

FIG. 11D illustrates the device of FIG. 11C wherein waste is beingexpelled from the device.

FIG. 11E illustrates the device of FIG. 11C wherein cells are beingdelivered from the device.

FIG. 11F illustrates another embodiment of a device of the presentinvention adapted to effect cell washing and/or buffer replacement andcell injection wherein buffer is being drawn into the device.

FIG. 11G illustrates another embodiment of a device of the presentinvention adapted to effect cell washing and/or buffer replacement andcell injection wherein buffer is being drawn into the device.

FIG. 11H illustrates the device of FIG. 11G in which the cell chamber isin operative connection with the housing or cylinder.

FIG. 11I illustrates embodiment of a plunger system operable to effectwashing or buffer replacement in a standard vial such as a cryovial.

FIG. 12 illustrates a cell washing and/or buffer replacement system ofthe present invention that can effect multiple dilutions and gravitysedimentation to, for example, remove transport or hibernationsolution/medium from the cells prior to injection.

FIG. 13A illustrates an embodiment of an integrated transport, wash,buffer replacement and delivery system of the present invention.

FIG. 13B illustrates several details of the flow path of the system ofthe FIG. 13B.

FIG. 13C illustrates position of the system of FIG. 13A so that asyringe thereof is vertical to allow settling.

FIG. 13D illustrates the system of FIG. 13A positioned so that cellsremain at a neck or outflow of the syringe for delivery.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 2 sets forth several embodiments of systems of the presentinvention for use in delivery of an injectate or injection fluid, andparticularly an injection fluid containing cells, to a brain of apatient. FIG. 3 sets forth several embodiments of systems of the presentinvention for delivery of an injection fluid, and particularly cells, tothe heart of a patient. Several embodiments of the present invention arediscussed below in detail with respect to delivery of cells to the brainor external heart of a patient. However, one skilled in the artappreciates that the devices, systems and methods of the presentinvention can be used to deliver many different types of substances tomany different tissues, internal to the body as well as to the skin.Moreover, the devices, systems and methods of the present invention areapplicable to open surgery or endoscopic needle-based deliveries as wellas to catheter-based deliveries.

The systems of FIGS. 2 and 3 are similar in overall architecture andoperation, and the systems of the present invention will be describedgenerally with reference to FIG. 3 and with respect to the delivery ofcells through the outer surface to the tissue of the heart.

In general, cell therapies are believed to work by replacing diseased ordysfunctional cells with healthy, functioning ones. However, themechanisms of the therapies are not well understood. As described above,therapeutic treatment involves harvesting cells from the body (such asadult stem cells) and later implants such cells. As discussed above, thetechniques are being applied to a wide range of human diseases,including many types of cancer, neurological diseases such asParkinson's and Lou Gehrig's disease, spinal cord injuries, and heartdisease. Many factors are considered when selecting an autologous or anallogeneic stem cell transplant. In general, autologous stem celltransplants (since the donor and the recipient are the same person andno immunological differences exist) are safer and simpler thanallogeneic (donor cells from a healthy donor other than the recipient)stem cell transplant. However, allogenic cells can be bettercharacterized and controlled.

FIG. 3 illustrates, for example, the harvesting of autologous bonemarrow cells or other cells from a patient 10 using a harvesting devicesuch as syringe 20 before or during an injection procedure. Theharvesting of bone marrow cells from the thigh of a patient isdiscussed, for example, in U.S. Pat. Nos. 6,595,979 and 6,835,193, thedisclosure of which are incorporated herein by reference. Suchautologous cells or allogenic cells from a donor can be placed in a cellstorage container or facility 30 for use at a later time, which may forexample, include incubation, concentration and freezing of the cellsand/or other processing. Shortly before delivery to a patient, cells canbe removed from cell storage 30 for processing in a cell processing unit40 and/or other units before delivery (for example, thawing and otherprocessing). Autologous cells can also be harvested and relativelyquickly delivered to a patient with or without substantial interveningprocessing.

In several embodiments of the present invention, cells are delivered toa container 50 (for example, a syringe) in a carrier fluid as known inthe art. Cells can also be harvested directly into container 50 from thepatient. The contents of container 50 are preferably pressurizable forinjection into the tissue of a patient. Prior to delivery of thecell-containing fluid to container 50, measurements relative toeffective delivery of cells to heart or other tissue can be made usingone or more inline measuring units or systems 70. Measuring unit 70 can,for example, measure cell count, cell viability, injection fluiddensity, temperature, nutrient level, gas level, composition etc. InFIG. 3, container 50 is illustrated as being connected to a poweredpump/injector system 100 which is operable, for example, to pressurizethe contents of container 50 for injection into the tissue of thepatient. Using, for example, connection mechanisms known in the art,container 50 (for example, a syringe) can be removably connectable topowered pump/injector system 100. Harvesting device 20 can, for example,harvest cells directly into container 50 as described above, and anysubsequent storage and/or processing of cells can take place incontainer 50.

Measuring unit 70 and or other measuring unit(s) or system(s) 72 canremain in operative connection with container 50 while container 50 isoperatively connected to pump/injector system 100 to continue to monitorthe state of the injection fluid prior to and during injection.Moreover, one or more maintenance units or systems 80 can be placed inoperative connection with container 50 while container 50 is inoperative connection with pump/injector system 100 to maintain cells ina desirable state. For example, the injection fluid in container 50 canbe agitated to maintain the injection fluid in a generally homogeneousstate. The agitation of a multi-component fluid is discussed inPublished PCT International Patent Application Nos. WO 00/53096, WO00/53242, WO 00/64353, WO 03/053494, WO 03/053554 and WO 03/095000, thedisclosures of which are incorporated herein by reference. Moreover, theviability of cells can be maintained by maintenance unit 80. Forexample, temperature, pH, pressure, nutrients, gases etc can bemaintained within desirable ranges and waste can be removed. Variousaspects of cell maintenance are discussed, for example, in U.S. Pat. No.6,758,828, the disclosure of which is incorporated herein by reference.

Each of the various systems or units of the present invention can, forexample, be in unidirectional or bidirectional communication with acontrol system 200 that can, for example, include one or more controlunits or controllers including one or more processors or microprocessors200, which (as known in the control arts) can include one or moreprocessing units 212 and associated memory storage units 214. Controlsystem 200 can be centralize or distributed within system 5. Asillustrated in FIG. 3, feedback or closed loop communication paths can,for example, be used to control the various components of system 5before, during and after an injection procedure to control the variouscomponents of system 5. Moreover, communication between facility (forexample, hospital) information systems and system 5 can be provided via,for example, control system 200.

As also illustrated in FIG. 3, more than one container (for example,syringe) can be placed in operative connection with pump/injector system100 to inject more than one fluid in the tissue of patient 10. In FIG.3, three containers 50, 52 and 54 are illustrated, but less than or morethan three containers and associated fluids (which may contain liquid,solid and/or gaseous components) can be provided. Many type ofadditional fluids including, but not limited to, flushing or diluentsfluids such as saline, viscosity adjusting fluids, imaging contrastfluids, and/or nutrient fluids, can be provided. The flow of fluid fromvarious pressurizable containers can, for example, be controlled via amanifold system 90 in fluid connection with containers 50, 52 and 54 andin communicative connection with control system 200. One or more ports92 can be provided, for example, in manifold system 90 to provide forfluid connection to other fluid sources which can include on or moreother powered pump/injector systems 94 and/or one or more manuallyoperated syringes 96. Manifold system 90 can also include one or moreports through which waste (which may present a biohazard) can betransmitted to an appropriate waste container 98. Manifold system 90 canfurther include or be in fluid communication with one or more mixers ormixing systems 99 to, for example, effect mixing of one or more fluids.

Injection fluid is delivered from manifold system 90 (or directly fromcontainer 50 and other containers in case of a system in which manifoldsystem 90 is absent) through one or more fluid path elements 310 (forexample, flexible tubing), each of which can include one or more lumens,to a patient interface 400 (for example, a needle or a catheter) forinjection into the patient's tissue. One or more measurement units orsystems 74 can be provided in connection with fluid path element 310 orin connection with patient interface 400 for measurement of variousvariables including fluid flow rate, fluid pressure, fluid density, cellcount, cell viability, cell maintenance variables etc. Such informationcan, for example, be transmitted to controls system 200 and theoperation of system components including, for example, pump system 100,cell maintenance unit or system 80, manifold 90 and patient interface400 can be controlled, at least in part, on the basis of such data orinformation. System 5 can further include a patient interfacepositioning control system 460 which can operate to facilitate manualpositioning or to partially or fully automate the positioning of patientinterface 400.

Various other components or systems can be used in connection with thepresent invention. For example, one or more imaging devices or system(s)500 (for example, X-ray systems (including, for example, angiography,venography and urography), computed tomography (CT) systems, magneticresonance imaging (MRI) systems, ultrasonic imaging systems, light basedimaging systems, and positron emission tomography (PET) systems) can beused in connection with the present invention. Imaging systems 500 can,for example, be used to track the position and viability of previouslytagged cells which are tagged with a marker that is detectible usingimaging system 500, to track the position of patient interface 400 or tomonitor one or more patient organs. Likewise, one or more physiologicalparameter monitors or monitoring systems 600 can be provided to monitorpatient physiological parameters including, but not limited to, cardiacfunction, respiration, blood oxygen level, and blood pressure. Data frommonitor(s) 600 can be provided to control system 200 and can be used incontrolling the operation of one or more of the components of system 5.Monitor(s) 600 can also be used to simply monitor the state of patient10 and ensure that the injection procedure does not harm patient 10.

System 5 can also includes a user interface system 700 that can, forexample, be used to provide user input and/or control into system 5 aswell as to provide information (for example, using visual, audibleand/or tactile indicators) to the user(s).

Details of various embodiments of a number of the components of and theoperative connection of such components within system 5 are set forthbelow. One skilled in the art appreciates that the various components ofthe systems of the present invention can be arranged or operativelyconnected in various manners and that various systems of the presentinvention need not include all of the components set forth in FIG. 2and/or FIG. 3.

Although headings and subheading are provided in the text of theapplication for organizational purposes, one skilled in the art willappreciate that concepts discussed under one heading or subheading canhave applicability in other headings or subheadings and the use ofheadings and subheading is not meant to limit the invention in anymanner.

Patient Interface

In general, patient interface 400 is the component of cell deliverysystem 5 that interfaces, interacts or interconnects with the patient todeliver a substance to the patient. Patient interface 400 is, forexample, shown in operative connection with the patient's heart in FIG.3 (and in connection with the patient's head/brain in FIG. 2). In ageneralized embodiment as illustrated in FIG. 4, patient interface 400includes one or more effectors 421 a, 421 b . . . 421 n, which canoptionally be moved or otherwise altered by one or more actuators 431 a,431 b . . . 431 n in operative connection with effectors 421 a, 421 b .. . 421 n. Fluid is brought to effectors 421 a, 421 b . . . 421 nthrough fluid path elements such as conduits 310 a, 310 b . . . 310 n.Actuators 431 a, 431 b . . . 431 n (and effectors 421 a, 421 b . . . 421n) are in communicative connection with control system 200. One or moresensors 441 a, 441 b . . . 441 n can be in operative connection witheffectors 421 a, 421 b . . . 421 n and in communicative connection withcontrol system 200 to provide, for example, feedback control ofeffectors 421 a, 421 b . . . 421 n. Such communication can, for example,be effected via a sensor interface 450, which can be in communicationwith sensors 441 a, 441 b . . . 441 n and control system 200. Sensorinterface 450 can also be integrated with control system 200. Operationof actuators 431 a, 431 b . . . 431 and effectors 421 a, 421 b . . . 421n can also be controlled, at least in part, on the basis of dataprovided by other systems sensors and monitors (for example, measuringunits 70, 72 and/or 74 and physiological parameter monitor(s) 600).

In current manual systems, there is a single effector—a needle, (or acatheter) a single piece of tubing connecting the needle to an injectionfluid source and no actuator connected to a control system. Theinterface positioning system is generally a needle grip or forceps usedby the doctor to manually maneuver the needle.

In one embodiment of the present invention, as discussed further below,one effector can be a single lumen needle or catheter and a secondeffector can be a depth stop mechanism. A fluid path element can be asingle piece of tubing in this embodiment and there may be no actuatorsin operative connection with the control system. In a more sophisticatedembodiment of the present invention, as discussed in more detail below,there can be a multi lumen (for example, concentric lumens) needle orcatheter with multiple fluid path elements in fluid connectiontherewith. The depth stop can be operated by an actuator. Anotheractuator such as a grip, ball screw, and motor can, for example, causethe needle to be withdrawn as the injectate is deposited into thetissue.

Fluid Path and Fluid Flow

A. Cell Protection and Viability in Fluid Path Elements

In general, any component with which the injection fluid comes intocontact during the injection procedure is considered part of the fluidpath. With reference to FIG. 3, for example, in the fluid deliverystage, the fluid path for the injection fluid (including, for example,cells) can, for example, include container 50, manifold system 90,mixing system 99, conduit 310, fluid contacting portions of inlinemeasurement unit or system 74 (if any), patient interface 400 and anyintervening conduits of connectors.

Within the fluid path (in the fluid delivery state or elsewhere—forexample, in the cell harvesting, cell storage, cell processing or anyintermediate stages) turbulent stresses contribute strongly tomechanical trauma of cells. Conditions that contribute to or promoteturbulence include wall irregularities, abrupt changes in tubedimensions, and disturbed flow upstream of a region of interest arecommon in current practice, as illustrated in the luer connector in FIG.1B. In this invention, cell damage resulting from hydrodynamic forcesduring handling and delivery of injection fluid are preferably minimizedby reducing the occurrence of or eliminating such conditions to, forexample, improve therapeutic value. However, even damaged or dead cellsmy have therapeutic value in some instances, such as myocardialregeneration, whereas dead cells appear to have no value in otherinstances, such as the treatment of Parkinson's disease.

Hydrodynamic forces can, for example, be produced by providing forgradual transition within and between all fluid path elements. Forexample, FIG. 5A provides an example of a relatively gradual transitionfrom a large radius section 810 to a smaller radius section 812. Alledges or corners are preferably rounded or radiused as illustrated inFIG. 5B, in which a first section 820 is connected to a second section822 via a connector 824 providing for radiused or rounded edges. In anyarea in which two fluid path elements are joined, the joints arepreferably butted to reduce or eliminates sharp transitions or toprovide for smooth internal diameters. For example, FIG. 5C illustratesthe use of an intervening fitting 834 to create a smooth transitionbetween tubing 830 and a needle 832. FIG. 5D illustrates the use ofanother fitting, transition or connector 844 connecting a first section840 and a second section 844 to provide for smooth internal diameters inthe fluid path. To ease the need to have very tight manufacturingtolerances, it is preferable that the fitting be relatively elastic sothat it can accommodate the variations in ID and OD of the parts beingmated. Alternatively, one of the other fluid path elements can berelatively more flexible and so adapt to the variations in the fitting.Assembly of one or more of the parts via insert molding can provideadvantages because the variations can be accommodated in the moldingprocess.

In most medical applications for the injection of fluid, tubing setshave no specific requirements other than containing system pressurewithout leaking and compatibility with the injection fluids. However, incertain applications that have more specific requirements including, butnot limited to, cell delivery, delivery of ultrasound contrast anddelivery of nuclear medicine, current tubing sets and connectors for usetherewith (for example, Luer fittings) have serious shortfalls.

As described herein, in the case of delivery of cells, there issensitivity to shear stresses induced in the cells. Moreover, there issensitivity to lost volume (as relatively small volumes are delivered).Further, trapped material left in a connector can present a biohazard.Similarly, in delivery of ultrasound contrast there is sensitivity tolost volume as small volumes are typically delivered. Moreover, standardor conventional fittings used in the industry have areas where bubblescan collect and not be delivered to the patient. Nuclear medicine alsouses relatively small volumes. Moreover, any trapped material left in aconnector presents a radioactive hazard.

To limit loss, it is desirable to use the smallest diameter of tubingpossible. In the case of cell delivery, however, care must be taken toavoid excessive shear. Currently most low-pressure tubing sets have abore diameter on the order of approximately 0.060 inches. For certainapplications the tube diameter can be on the order of approximately0.020-inch diameter. This reduction in diameter reduces volumetric lossand increases flow velocity to assist in prevention of adherence ofcells (or bubbles etc.) on the walls of the tubing. The length of thetubing is also preferably minimized.

Currently, luer fitting are widely used as connectors in connection withmedical tubing sets and other medical components. The design of luerfittings cause the formation of small volumes of fluid that are not inthe direct fluid path. That is, there are small volumes in the luerconnector wherein material can collect and not be removed by a flush.These common luer fittings are not designed to maintain constant uniformdiameter throughout the system.

As illustrated, for example, in FIGS. 5E through 5H, the presentinvention provides a number of other fittings or connectors that providefor relatively low fluid loss. Such connectors also preferably providefor smooth transition between fluid path elements to reduce turbulence.In several embodiments of fittings or connectors of the presentinvention, a face seal can be used. In such an embodiment, the fittingincludes flat faces that are mated together to make a seal. The facescan include compressible sealing elements. To reduce the likelihood ofleaking, an annular seal (such as an o-ring) can be used. Use of anannular seal can provide for use of the connector at relatively highpressures with little tightening torque.

In the embodiment of FIG. 5E, a first tubing section 910 (for example, asmall diameter tubing section) is connected to or terminated by a malefitting 920. Male fitting 920 includes, for example, a connectionmechanism such as threading 922. An end 924 of male fitting 920 includes(or has in operative connection therewith) a sealing member such as anO-ring 926. A second tubing section 930 (for example, a small diametertubing section) is connected to or terminated by a female fitting 940.Female fitting 940 includes, for example, a cooperating connectionmechanism such as cooperating threading 942. As illustrated in the leftside of FIG. 5E, preferably there is no significant area change orchange in inner diameter upon connection of male fitting 920 and femalefitting 940.

In the embodiment of FIG. 5F, a first tubing section 910 a (for example,a small diameter tubing section) is connected to or terminated by a malefitting 920 a. Male fitting 920 a includes, for example, a connectionmechanism such as threading 922 a. An end 924 a of male fitting 920 a isangled or tapered. A second tubing section 930 a (for example, a smalldiameter tubing section) is connected to or terminated by a femalefitting 940 a. Female fitting 940 a includes, for example, a cooperatingconnection mechanism such as cooperating threading 942 a. Female fitting940 a further includes a seating 944 a adapted to seat tapered end 924 aof male fitting 920 a. Seating 944 a can, for example, have a taperangle generally the same as or slightly greater than the taper angle oftapered end 924 a of male fitting 920 a. As illustrated in the left sideof FIG. 5G, preferably there is no significant area change or change ininner diameter upon connection of male fitting 920 a and female fitting940 a. Male connector 920 a and female connector 940 a (or portionsthereof) can be formed of a resilient or somewhat compliant material toassist in forming a sealed connection.

FIG. 5G, on the right side thereof, illustrates standard luer connector1 (as illustrated in FIG. 1B) in a disconnected state and in a connectedstate. A cross-section of the fluid path created upon connection of maleluer connector 1 a and female luer connector 1 b, clearly showing theresultant lost volume region 1 c and resultant sharp transitions, isalso illustrated. For comparison, the left side of FIG. 5G illustratesthe male connector 920 a and female connector 940 a of FIG. 5F in aconnected state as well as a cross-section of the resultant fluid path,illustrating that there is no lost volume region or sharp transitions ininner diameter.

In the embodiment of FIG. 5H, a first tubing section 910 b (for example,a small diameter tubing section) is connected to or terminated by a malefitting 920 b. Male fitting 920 b includes, for example, a connectionmechanism such as threading 922 a. Male fitting 920 b further includesand extending end member 924 b. A second tubing section 930 b (forexample, a small diameter tubing section) is connected to or terminatedby a female fitting 940 b. Female fitting 940 b includes, for example, acooperating connection mechanism such as cooperating threading 942 b.Female fitting 940 a further includes a seating 944 b adapted to seatextending end member 924 b of male fitting 920 b. Seating 944 b can, forexample, including an extending member 946 b adapted to mate withextending member 924 b of male fitting 920 b. Seating 944 b furtherincludes a flexible sealing member 948 b (for example, an elastomericsleeve member) to encompass and assist in forming a sealing connectionof extending members 924 b and 946 b. As illustrated in the left side ofFIG. 5H, preferably there is no significant area change or change ininner diameter upon connection of male fitting 920 b and female fitting940 b.

FIG. 5I illustrates an embodiment of a luer-type fitting or connector950 having a through bore 952 sized to match the outside diameter oftubing 960 connected to fitting 950. Tubing 960 can, for example, beglued into luer-type fitting 950 in a position that allows a front face962 of tubing 960 to compress and seal against a front face 972 of asyringe 970 (or other flow path element). This compressing abutmentprevents fluid from entering a dead space area 954 of fitting 950.Fitting 950 further includes a tapered female portion 956 that mateswith a tapered male portion 974 of syringe 970.

FIG. 5J illustrates an embodiment of a luer-type fitting or connector950 a having a sliding component 952 a. A seal is made at a face 954 aof sliding component 952 a and tip or front face 972 of syringe 970 (orother flow path element). No seal is made at the luer tapers in thisembodiment. A smooth transition is provided for the fluid path fromsyringe 970 to sliding component 952 a. A smooth transition is alsoprovided for the fluid path from sliding component 952 a to tube 960 aconnected to fitting 950 a. In the illustrated embodiment, an O-ring orother biasing member 956 a at the rear of sliding component 952 aprovides spring compression or biasing force to hold face 954 a ofsliding component 952 a against syringe face 972.

FIG. 5K illustrates another embodiment of a luer-type fitting orconnector 950 b which provides functionality similar to that provided byfitting 950 a of FIG. 5J, but with no moving parts. A seal is made at aface 954 b on the interior of luer-type fitting 950 b and tip face 972of syringe 970 (or other fluid path element). No seal is made at theluer tapers. A smooth transition is provided for the fluid path fromsyringe 970 to luer fitting interior face 954 b. A smooth or gradualtransition is also provided for the fluid path from luer-type fitting950 b to tube 960 b. In that regard, the flow path within fitting 950 bincludes a tapered or angled region 956 b having a first diameterapproximately equal to the inner diameter of tube 960 b and a secondinner diameter approximately equal to the inner diameter of the syringetip opening. A swivel nut (not shown in FIG. 5K, but see FIG. 5J) on thesyringe tightens onto luer threads pulling face 954 b of luer-typefitting 950 b and syringe face 972 together and creating a seal.

FIG. 5L illustrates another embodiment of luer-type fitting or connector950 c having an internal protrusion or extending member 952 c that fitswithin the inner diameter of the syringe tip fluid path (or other fluidpath element). A smooth or gradual transition is provided by tapered orangled region or section 956 c within protrusion 952 c for the fluidpath from syringe 970 to luer-type fitting interior face 954 b. A smoothtransition is also provided for the fluid path from luer-type fitting950 c to tube 960 c via tapered region 956 c. In that regard, tapered orangled region 956 c has a first diameter approximately equal to theinner diameter of tube 960 b and a second inner diameter approximatelyequal (or slightly smaller than) to the inner diameter of the innerdiameter of the syringe tip opening. A seal is created via standard luertaper.

In another embodiment, two mating tapered elements are used. The taperedelements preferably have a greater angle of taper than a Luer connection(that is, greater than approximately 6 deg). In several embodiments, thetaper is in excess of 25 degrees. For example, in one embodiment a taperon the order of 45 deg can be used. The male part of the taper caninclude a smaller angle of taper than the female taper, (for example,about 5 degrees). The difference in taper allows contact in the centerover a small area to provide a reliable seal with relatively littletightening torque.

B. Fluid Path and System Capacitance/Delivery Efficiency.

In the delivery of, for example, stem cells into tissue such as theheart muscle or the brain, it can be desirable to deliver a sharp bolusof cells in, for example, ten or more locations. Efficient transfer ofcells to the muscle or the brain is important because of the limitedquantity of cells available. The pressure required to deliver a bolusmight not be available if there is too much capacitance in the system.Capacitance can defined as the ability of the system or an element ofthe system to increase in volume during pressurization, and then torelax to normal after pressurization. System capacitance can work like aspring absorbing pressure and releasing it when the pressure orrestriction on the other side (increase load from heart muscle) of theneedle is removed. The absorbing of pressure and subsequent release iswhy a system with a lot of capacitance will continue to deliver fluid ordrip when a needle is withdrawn from the injection site. This drippingdecreases the efficiency of the cells delivered, for example, to theheart and cells can be leaked into undesirable locations.

A certain level of capacitance in a fluid delivery system may bedesirable, however, in certain circumstances. For example, if cells aredamaged at a known shear force, the system can be designed to haveenough capacitance to prevent the pressure from rising to the level thatwould cause shear to occur in the cells.

However, excessive capacitance is undesirable. Capacitance reductioncan, for example, be accomplished in several ways: As set forth in FIG.6A, a braided or otherwise “stiffened” (for example, having thickenedwall) connector tubing 1010 connector tubing can be used. Thefabrication material for container or syringe 1012 of FIG. 6A and otherfluid path elements can be chosen to be stiff (for example,polycarbonate can be chosen rather than, for example, polypropylene—aspolypropylene expands more under pressure). Alternatively oradditionally, the wall thickness of syringe 1012 can be increased.O-rings 1014 or other sealing rings can be used around the perimeter ofsyringe plunger 1016 rather than elastomeric plunger covers. In thatregard, elastomeric (for example, rubber) plunger covers can flexresulting in increased capacitance. Teflon seals can also be used in thesyringe plunger. A tight fit of syringe flange 1018 in injector system1020 can be provided, or a syringe sensor can spring load the flangeforward (instead of rearward) as represented by element 1024 in FIG. 6A.Air causes capacitance because of its compressibility, therefore theefficiency of air removal from the system can also be improved. Forexample, a membrane that allows air to pass but prevents fluid frompassing can be provided. Such a membrane can, for example, work at lowpressure provided that the pressure is less than the membrane breakdownpressure of the filter. As represented by designation 1026, backlash canbe removed from system wherever possible. For example, the plunger canbe spring loaded to reduce or eliminate backlash.

It may be also desirable to control the amount of capacitance of asystem to protect the cells from exposure to damaging shear forces. Ifstem cells are destroyed at a know shear force, one can determine whatpressure will develop that shear force for a known system configuration,i.e. if the disposable set is defined and a maximum shear force isestablished, then a maximum system pressure can be determined to reduceor eliminate the chance of exceeding the maximum shear force. As setforth in FIG. 6B, the system can include silicone tubing or othercompliant tubing 1010 b that allows only a defined amount of pressure tobe delivered before it yields (for example, causing it to bulge) andlimits the pressure. A maximum pressure setting can be set on aninjector or pump system such as system 100 which can, for example,deliver to the maximum pressure and hold that pressure. As illustratedin FIG. 6C, a separate spring loaded vessel (for example, a syringe 1030c including a plunger 1032 c loaded by a spring 1034 c retained withinsyringe 1030 c with, for example, a mechanical abutment or stop 1036 con the rearward end of syringe 1030 c) can be provided to absorbpressure and to provide an indication to the operator that the desiredpressure has been exceeded. This can, for example, be useful for theoperator to determine when it is safe to remove the needle from theheart, brain or other tissue. If the pressure spikes and spring loadedplunger/indicator 1034 c moves, then the operator can hold the device inthe tissue until the pressure drops and spring loaded plunger 1034 creturns to its original state (indicating that all the cells have beendelivered to the tissue).

FIG. 6D illustrates a syringe 1040 including spring isolated plunger1042 designed such that an operatively connected spring or other biasingmember 1044 will not compress under a predefined threshold load.However, if pressure in syringe 1040 exceeds a threshold, spring 1044will compress, limiting the pressure that can be developed in syringe1044.

As illustrated in FIG. 6E, a normally closed, a push-button valve can beprovided to activate pump system such as pump or injector system 100 andopen fluid passage to needle 400: A normally closed push-button valvebetween the container/syringe 50 and the patient interface/needle 400can, for example, have an electrical switch to initiate the pump system100. By pressing or otherwise activating this valve, the fluid passageto the needle is opened and an electrical switch triggers injector/pumpsystem 100. When the button is released, injector 100 can, for example,stop and the valve close. If the button is held down, injector 100 stopsat a predetermined volume. Any dripping from the tip of needle 400 as aresult of system capacitance is eliminated because the valve is closedand will not allow fluid to pass to needle 400. This embodiment canreduce the amount of wasted cells.

FIGS. 6F and 6G illustrate the use of a one-way check valve 1052,respectively, in a needle 1050, respectively. When the needle is placedin tissue (see FIG. 6F) such as in the heart, an activation rod 1054,respectively, is pushed rearward and check valve 1052, respectively, isopened. When needle 1050 is withdrawn from the tissue (see FIG. 6G),activation rod returns 1054 to a relaxed or unstressed state and checkvalve 1052 is closed, thereby preventing fluid leakage.

FIG. 6H illustrates reduction in capacitance by reducing the totalvolume of the system (as in comparing the injection system of the rightside of FIG. 6H with that of the left side thereof). For example, thesize of container/syringe 50 can be reduced and the length of fluid path310 can be shortened. FIG. 6I illustrates the use of increase wallthickness (in for example, two syringe 50 and 50 a) to reduce fluid pathelement capacitance.

FIGS. 6J through 6N illustrate an embodiment of a syringe 1060 in whichcapacitance is substantially reduced or eliminated. As described above,capacitance can negatively impact fluid delivery precision. Excessivecapacitance delays fluid movement while the system expands as a resultof hydraulic pressure. Subsequently, at the end of the injection, fluidwill slowly exit or dribble while the system deflates as a result ofpressure loss. This expansion and deflation prevents precise andcontrolled delivery. Specifically, with cell delivery, uncontrolledcapacitance causes a slow continuation of exudates to exit the devicewithout the proper force to deliver the cells to the target tissue. Inthe extreme case the exudates may even continue after the device isremoved from the tissue, potentially exposing other, non-target tissueor clinicians to an unsafe condition.

In the embodiment of FIGS. 6J through 6N, the syringe barrel is designedfor minimum strain or radial defection during the maximumpressurization. This can, for example, be accomplished by appropriatematerial selection and dimensioning as discussed above. Tensilestrength, modulus of elasticity, and environmental conditions areimportant characteristics. As an example, in one embodiment usingpolycarbonate and given a radial stress of 115 psi, the maximum radialdeflection is 0.001 inches, corresponding to a total volume increase of0.10 mL within a 17 mL total volume syringe. Use of a solid,non-elasotmeric plunger 1062 with, for example, an O-ring side sealingmember 1064 also helps minimize capacitance. Such a plunger minimizesfluid contact with an elastic surface (as, for example, compared to aplunger with an elastomeric cover of the forward and side surfacesthereof), while providing a seal against leakage. As described above,delivery tube can also designed and constructed to minimize capacitance.Syringe 1060 of FIGS. 6J through 6N, is preferably fabricated from anoptically clear polymer such as polycarbonate to ensure visibility ofthe fluid contents. The material of syringe 1060 is also preferablyblood- and cell-contact compatible. Optionally, the internal aspects ofthe fluid path and fluid path elements (including syringe 1060 and otherfluid path elements) may have a lubricious coating, such as HYDROMER® (ahydrogel material made by the interaction of poly-vinylpyrrolidone withone of several, isocyanate prepolymers) available from Hydromer, Inc. ofBranchburg, N.J. to, for example, reduce friction and/or maintain cellviability. Coatings can also be used to reduce plating or walladherence. The internal aspects of the barrel are preferably adapted tominimize fluid turbulence and cell viability by, for example, providingradii at diameter transitions, as well as non-acute angles. A connectorsuch as a male luer connector or fitting or a fitting of the presentinvention as described herein can be provided incorporating a rotatingnut to aid in attaching the disposable tubing.

A distal angle of a forward section 1066 of plunger 1062 can be slightlysmaller than the distal angle of a transition region 1068 of the syringebarrel (see FIG. 6J). This angle mismatch provides a channel for cellsto exude through distal opening or syringe tip 1070 without gettingtrapped against the interior barrel angle. Typically, conventionalsyringe designs incorporate matching angles between the plunger andbarrel. However, this arrangement causes both surfaces to touchsimultaneously over a substantial portion thereof. While, such anglematching is a good design for most non-viable fluids, angle matchingcould damage cells caught between the two surfaces and lower the overalleffectiveness of cell therapy. The described mismatch preferablyminimizes the amount of residual fluid, while maintaining cellviability. One or more other abutment elements 1072 (see FIG. 6J) canadditionally or alternatively be used to prevent mating of a forwardsurface of the plunger over a substantial area thereof with a surface ofthe transition region of the syringe.

When injecting into tissue with a system having capacitance, thepressure can ramp up quickly and gradually drop as the capacitance istaken up and fluid is injected into tissue (for example, the heart orthe brain—see FIG. 6O). Once needle 400 is removed from the tissue, thepressure will drop suddenly as the tissue restriction to flow has beenremoved. When the pressure drops (as, for example, sensed by a pressuresensor 51′), the drive of pump system 100 can be reversed (for example,by moving a drive member in operative connection with a plunger incontainer/syringe 50 in a rearward direction), thereby stopping fluidfrom leaking out of patient interface/needle 400. Pressure sensing ininjector systems is, for example, discussed in U.S. Pat. Nos. 6,673,033,6,520,930 6,488,661, 5,808,203 and PCT International Patent ApplicationPublication No. WO 00/06233, the disclosures of which are incorporatedherein by reference. Pump system or injector 100 can be programmed toreverse or retract a certain amount at the end of an injection torelieve residual pressure from the system capacitance. FIG. 6Pillustrates a graphical representation of a pressure profile with needle400 within the tissue. FIG. 6Q illustrates a pressure profile withsystem capacitance and needle 400 removed from the tissue at time t.

C. Fluid Viscosity

In the case of cell therapy, the injectate or injection fluid typicallyincludes at least one cellular component and a liquid or carriercomponent. Preferably the cellular component includes live, andundamaged cells, but damaged cells as well as dead cells can havetherapeutic value also. The viscosity of the injectate can varysignificantly. As discussed briefly above, cells (and cells supported,for example, on microspheres) do not behave like uniformly dispersedparticles in a fluid. Among the problems arising in the fluid transportof cells are tendencies to clump together, to settle, to plate or adhereto walls of the fluid path and/or to stay in place while liquid flowsthrough the “packed” cells (if flow velocities are sufficiently low). Anumber of approaches to address these problems are discussed above.

As also briefly discussed above, in several embodiments of the presentinvention viscosity can be used to reduce the significance of orovercome one or more of the problems listed above as well as otherproblems. Blood is a non Newtonian fluid, meaning that the viscosity isa function of flow velocity and thus the conditions of measurement.Blood at a normal hematocrit has a viscosity of about 4 centipoise at37° C. under common ex-vivo test conditions. The viscosity of water isapproximately 1 centipoise at 20° C. The viscosity of plasma is inbetween the viscosity of blood and the viscosity of water. A significantcomponent of plasma is albumin, a large protein, which partly explainswhy plasma's viscosity is greater than that of water. As theconcentration of albumin is increased, the plasma becomes more viscous.

If the fluid in the injectate is increased in viscosity, severalbenefits are realized. For example, the cells will tend to settle moreslowly. If the density is increased at the same time, the tendency tosettle will be decreased as well. As a result, for example, little or noagitation may be required to maintain the injectate in a homogeneousstate. With regard to flow characteristics, a greater force is generallyrequired to pull the cells off the walls and to break up the clumps orpacking of the cells. With a more viscous fluid, the pressure at theinjection site (commonly the tip of patient interface/needle 400) willbe much greater before it starts leaking back the needle track, causingmore of a cavity to be created in the tissue for deposition ofinjectate. Further, the more viscous injectate cannot as easily backflowor retrograde back up needle track or through fine structures/cavitiesin the tissue. In several embodiments of the present invention, it ispreferred that the injectate viscosity be greater than 4 centipoise and,more preferably, greater than 6 centipoise. However, the optimalviscosity will typically depend upon patient interface 400 (typically aneedle or a catheter system). In that regard, patient interface 400 ismost likely to be the fluid path component with the minimum innerdiameter. Given the flow characteristics of patient interface 400 andother system consideration, on skilled in the art can readily determinean optimal viscosity for a given application.

The viscosity of the injectate can be increased in several ways. One wayof increasing viscosity is to increase the fraction of cells in themixture. Increasing cell concentration has the additional benefit ofrequire a smaller injection volume to deliver a desired number of cells.Thus, less pressure is built up in the tissue, and there will be less ofa chance of fluid backflow or retrograde flow. Because of the increasednumber of cells per volume, the flow rate can also be reduced, therebyhelping to maintain the same shear strain in the fluid. The cells can beconcentrated by settling or centrifuging to create a concentratedfraction. Alternatively, the cells can be collected on a filter and backwashed or suctioned into the delivery system.

Alternatively, the viscosity of the carrier liquid can be increased withthe addition of, for example, non-essential or “excipient” cells (forexample red blood cells) or other particles, such as collagen particles,for example spheres, in the range of tens of nanometers to tens ofmicrons in diameter.

In the strategies discussed herein, it is desirable to maintain theproper osmotic pressure so that the cells are not adversely affected byswelling or shriveling. This can be measured and corrected by addingwater or a salt solution as appropriate. It is also necessary tomaintain the proper pH which can be done through various organic orinorganic buffers.

The viscosity of the injectate can also be increased by increasing theviscosity of the molecular part of the fluid, for example, by increasingthe fraction of albumin in the liquid. This result can be accomplishedby simply adding albumin to the fluid. Alternatively, the cells can beconcentrated and separated from much of the liquid as discussed above,and a new liquid having a sufficiently increased viscosity added. Theaddition of dilute collagen molecules is another alternative. Bothcollagen and albumin have the advantage of occurring naturally in thebody, and both are readily removed or decomposed. Other naturallyoccurring large molecules can be used as clear to those skilled in theart. Synthetic molecules can also be used. For example X-ray contrast isa large molecule, is water soluble, and has a high viscosity atphysiological osmolality. Among X-ray contrasts, the greatest viscositycomes from those with dimmeric molecules, for example Visipaque(iodixanol) manufactured by Amersham Health, a division of GeneralElectric Medical Systems. The 270 mgI/ml concentration has a viscosityof 6.3 centipoise at 37 centigrade, 12.7 centipoise at 20 centigrade anda physiological osmolality. The 320 mgI/ml concentration has a viscosityof 26.6 centipoise at 20 centigrade, also at a physiological osmolality.Thus, a reasonable amount of Visipaque will sufficiently increase theinjectate viscosity. Addition of an imaging contract can also assist ina marking, tracking or mapping function in conjunction with imagingdevice or system 500. Other suitable synthetic materials includesynthetic peptide hydrogels use to form the Puramatrix tissuescaffolding, made by 3DM Inc. of Cambridge, Mass. In sufficiently lowconcentrations, the long chain molecules increase viscosity of theinjectate, but do not form a solid gel. Synthetic infusion products suchas Hemohes, Gelofusine, and Venofundin manufactured by B Braun could beused. A particularly useful material is carboxymethylcellulose (CMC), anexample of which is Aqualon manufactured by Hercules, Inc. of WilmingtonDel. A 2% solution has a viscosity of 60-80 centipoise. CMC is used as aviscous carrier or excipient in Sculpta, an injectable treatment forlipoatrophy, available from Aventis Pharmaceuticals, Bridgewater, N.J.Additionally, molecules or droplets of inert synthetic large moleculessuch as perfluorocarbons or perfluoropolyethers (see, for example,Published PCT International Application No. WO002005072780A2, thedisclosure of which is incorporate herein by reference) can be used. Itis preferable to use molecules that are sufficiently large thatviscosity is increased quickly.

The viscosity of the injectate can optionally be increased to the pointthat it can be described as a gel or a paste. In the case of a gel orpaste, the cells move very little with respect to each other. The cellscan be considered to be trapped in the gel. When the gel is injected,there is very little backflow or retrograde of injectate back throughthe needle track or through the tissue. The cells would initially staywhere they were deposited. If the gel is made primarily of collagen,synthetic peptide hydrogels, or alginate, and the volume deposited issmall enough that oxygen and nutrients can diffuse to the cells (whichdepends upon the density and type of cells), the cells will eventuallybe freed by the body's decomposition or degradation of the gel. Thecells are then able to migrate, divide and/or perform the function(s)needed to achieve the treatment.

Because of the high viscosity of a gel or paste, the injections areeither relatively slow, or a lubricating fluid, for example water, canbe used between the gel and the walls of the fluid path to reduce thepressure and shear stress on the cells. Gels containing water tendnaturally to form a water layer near the fluid path wall. Alternately,water can be injected from one of multiple containers or syringes 52 and54 etc. concentrically around the gel as a lubricant. In the case of agel, it is important that, as discussed above, transitions in innerdiameter of fluid path elements be as gradual as possible.

The cells can, for example, be mixed with a precursor or pregel materialbefore gelling occurs. Alternatively, an open gel can be created andthen used as a filter to collect the cells. The cells would be embeddedinto the gel. Previous work on tissue scaffolds can be applied in thisway to cell injections. An example of such a matrix is the Puramatrixscaffold made by 3DM Inc. of Cambridge, Mass.

As the viscosity is increased still further, a rod or other element of“solid” cell-containing material is “injected” or deposited. The solidinjectate can be created or formed as a rod outside of the injectiondevice, and then loaded into the injection device as, for example, leadis loaded into a mechanical pencil. Alternatively, the injection devicecan be loaded with fluid/liquid injectate that solidifies in theinjection device. Components that form a gel can also be separatelyintroduced into the injection device, where they are mixed, and the gelforms. An example of such a material is alginate, which forms a gel inthe presence of calcium ions. A material of this type is made by NeuralIntervention Technologies, Inc of Ann Arbor, Mich. The alginate and thecells can, for example, be mixed. When calcium chloride is added, thecells are trapped in the alginate matrix as it forms. This solid canthen be injected and the cells and will not leak back the needle trackor elsewhere.

Alternately, a solid, cell-filled matrix can be created by growing cellsinto and through the matrix. Patient interface 400 in the form of aneedle can be filled with the matrix by simply inserting the needle intothe matrix and cutting a core. This coring/loading can be done by handbut is more repeatable if done using a mechanized fixture suitable toensure that the needle cuts different sections each time. When theneedle is placed into the proper position in the patient's tissue, thesolid core is displaced from the needle by, for example, pushing frombehind with liquid or with a solid stylet.

As mentioned above, one of the benefits of injecting a high-viscositymaterial or a solid is that it does not leak back the needle track, leakout of the tissue and into surrounding tissue/organs or spreadthroughout the tissue. However, those attributes limit the cell locationto a small area within the tissue. In some uses, such as cellimplantation in the scalp or into the brain for Parkinson's disease,this limitation is not a problem. In some other applications, such ascell therapy for the heart, current theory of operation dictates thatthe cells be applied over a range of tissue area. Thus, to spread thecells over a range of tissue, it is desirable that the viscous fluid orsolid be injected as the needle is being pulled back as described above.The cavity created by the needle is filled, or optionally overfilledwith injectate, rather than the return of the displaced tissue. If theneedle is inserted at a shallow angle with respect to the tissuesurface, this approach allows a large area to be treated even with avery viscous material. This concept can be used with multiple needleembodiments as described above. The coordination of the injection andneedle withdraw is preferably accomplished, at least in part, usingcontrol system 200.

Alternatively, solid injectates can be premaufactured into cylindersthat are inserted into multiple implantation needles as lead is placedinto mechanical pencils.

In still a further embodiment, solid rods containing cells can be formedto themselves pierce and embed within tissue such as heart or braintissue without the assistance of a needle or catheter. Multiple piercingor penetrating rods or other solid injection elements can, for example,be applied generally simultaneously or in a single application as anarray with an applicator. FIG. 6R illustrates an example of anapplicator 1080 including breakaway sections 1082 of solid injectatethat are sequentially embedded, fully or partially, in the tissue (seethe right side of FIG. 6R). The breakaway material can serve any numberof additional functions, including, but not limited to, acting as abuffer that protects the primary therapeutic material from damage, amechanism to push the primary therapeutic to further depth in thetissue, a cap for the therapy site, or as a color, radiological or othermarker to allow the user to keep track of the injection visually or witha secondary sensing or imaging device.

If the needle holds more solid injectate than is delivered in a singleinjection, it is desirable to stop the delivery before or as the needleleaves the tissue. This is best done if control system 200 operates theinjectate delivery and a depth stop. In this case the depth stop can besettable by control system 200. The user can, for example, set theinitial depth and the volume to be delivered. The needle is theninserted until the depth stop contacts the tissue. The injector is thenactivated. As the injection occurs, the depth stop is moved so that theneedle is controllably extracted as the injectate is delivered, whilethe user simply maintains contact between the depth stop and the tissue.User interface 700 can indicate when the injection is complete and theuser can move to the next site. The depth stop can be used to ensureproper depth of penetration. Such drive mechanism can readily becontrolled by control system 200 using control algorithm procedures asknown in the art.

D. Generalized Cell Delivery Flow Modes

The discussion of the multiple and various flow modes and embodiments ofthis invention can, for example, be described generally with referenceto FIGS. 7A through 7F. In FIG. 7A, three fluid path elements are showndiagrammatically as concentric cylinders A, B, and C, in this figure.The distal ends marked respectively with Ad, Bd, and Cd are the endsclosest to the patient. The effectors are the distal ends of the fluidpath elements that interact with the patient and the means for holdingor position the effector. A fluid path is determined by one or more ofthe fluid path elements. In FIG. 7A, fluid path 1 is inside fluid pathelement A. Fluid path 2 is inside fluid path element B and outside offluid path element A. Fluid path 3 is inside fluid path element C andoutside of fluid path element B. Fluid paths 1 and 2 communicate wherefluid path element A ends, and fluid paths 2 and 3 communicate whereelement B ends. This communication may be at or in the patient, or atsome point before reaching the patient.

A fluid path can be made up of one or more physical fluid path elements,which may be made of any of the many materials known to those skilled inthe medical device arts that can contain the fluid within them, eitherflowing or static, without contaminating the fluid. For example, theycan be a single rigid fluid path element, such as a metal needle. Theycan be flexible, such as plastic tubing or catheters. Some elements canbe rigid and others can be flexible. Or, a single fluid path can be madeup of multiple fluid path elements, such as a flexible fluid pathelement, piece of tubing, connected to a rigid fluid path element, suchas a needle.

The fluid path elements illustrated in FIGS. 7A to 7F are downstream ofthe powered pump and the manifold or their equivalents, if they are usedin the system. By fluid path element is meant any element that touchesthe fluid, including the pumps and manifolds not shown in FIGS. 7A to7F. The effectors of FIG. 4 that contact the fluid are included as fluidpath elements.

The simplest fluid path is shown in FIG. 7B. It has one fluid pathelement A and one fluid path 1. In this invention, there are a number offluids that can and will be transmitted via this fluid path. In themanual practice discussed in the background, the fluid path element A isa needle that is connected to a syringe for injection of a fluid withcells or drugs. In the prior art manual methodologies, the pump is thesyringe and the operator's hand, the control system is his or her brain,the fluid being injected is the therapeutic fluid containing cells, andthere is no manifold. The pump directly injects in to the fluid path 1.

In several embodiments, the present invention replaces the hand with oneor more mechanically or electrically powered pumps or controlledinjectors. If multiple injectors and/or a multi-container injector areused with a manifold or valve arrangement (see, for example, FIG. 3),then several fluids can be injected either sequentially or serially. Asan example, fluids used include the therapeutic injectate, and could forexample include one or more of saline or a similar physiological fluidfor priming the fluid path elements or flushing the therapeuticinjectate, a viscosity modifying agent, a lubrication agent, a tissuecracking or opening agent, a site marking agent, and a sealing or gluingagent to seal the site or fill the needle or catheter track. Thedetailed uses of such fluids are discussed in various parts of thisdescription.

One embodiment with two fluids includes the therapeutic fluid andsaline. The fluid path 1 is first filled with saline to remove all airfrom the path. Then, depending upon the volume contained in the fluidpath as compared to the volume of the therapeutic fluid to be injected,the purging fluid can be left in the fluid path while the effector,needle in this case, is placed in the target tissue. The purge fluid inthe fluid path is simply injected into the tissue before the injectionof the therapeutic fluid. And, optionally, the injection of thetherapeutic fluid can be followed by an injection of a flush fluid, forexample saline, to drive more of the therapeutic fluid out of the fluidpath elements and into the tissue.

In FIG. 7C, there are two fluid path elements, A & B. Similarly, thereare two fluid path elements shown in FIG. 7D. An arrangement similar tothis was disclosed in Published U.S. Patent Application No. 2004/0254525A1, the disclosure of which is incorporated herein by reference. In thatapplication, it was used to surround the injection of a drug that harmedvessel walls with a second fluid that did not harm the walls. In thecurrent invention, injecting the therapeutic injectate through fluidpath 1 and a fluid such as saline with a relatively lower viscositythrough fluid path 2 will help protect the cells in the therapeuticinjectate from the high shear stress as they travel through fluid path 2to the patient. Using readily available computational fluid dynamic(CFD) modeling, for example Fluent and other software packages availablefrom Fluent, Inc. of Lebanon, N.H., it will be possible to select theappropriate flow rates and velocities to maintain the desired flow andsafe shear stress levels. In this case, it is likely that the distanceof travel outside of fluid element A, the physical distance from Ad toBd, will be relatively long.

A second application of the fluid paths of FIGS. 7C and 7D involvesnon-simultaneous delivery of fluids. The initial fluid through fluidpath 2 could be relatively high pressure and velocity to “crack” or opena space in the tissue. After that fluid dissipates into the tissue, orafter it is sucked back out through fluid path 2, then the therapeuticinjectate can be delivered through fluid path 1. Alternatively, thesecond fluid is delivered through fluid path 2 after the therapeutic toflush or drive the therapeutic fluid into the tissue. A thirdapplication, show in FIG. 7D, has the vector distance Ad to Bd be veryshort or in fact negative. (A negative Ad to BD vector distance meansthat the fluid path element A actually sticks out past fluid pathelement B.) In this application of the embodiment, the injectate flowstoward the patient in fluid path 1, and some of the injectate is drawnback in away from the patient through fluid path 2. The laws of fluidmechanics cause particles or objects in a developed fluid flow ofsufficient velocity to concentrate in the center of the flow away fromthe walls. This happens in normal blood flow in the arteries. Inphysiological systems, some blood vessel branches use a “cushion” oftissue to take blood from the cell rich center flow. (Physiology andBiophysics of the Circulation, 2^(nd) Edition 1972, Alan C Burton, LC#70-182003, chapter 5) In the opposite way, fluid path 2 is removingsome of the fluid with less or no cells, so that the total fluid volumeinjected into the tissue is reduced for a given number of cells beingdelivered to the tissue. This reduces or eliminates the tissue swellingneedlessly increased by fluid that is of no therapeutic value.Alternatively, fluid path 2 could withdraw fluid just long enough toremove the purging fluid from the system, but not withdraw fluid whenthe therapeutic fluid arrives. Fluid path 2 could incorporate a filterto ensure that cells are not needlessly removed and wasted.

Alternatively, fluid path 1 could be used for delivery of thetherapeutic fluid and fluid path 2 is used to delivery a site markingfluid or a needle or catheter track filling. This is most likely donewith an embodiment where fluid path element A extends past the end offluid path element B so that fluid path 1 is not occluded by the trackfilling fluid. In addition, fluid path 2 could transport a fluid thatreacts with the fluid in fluid path 1, for example, calcium ions thatwill cause the alginate in fluid path 1 to gel.

One of the challenges with cell delivery is that the cells tend tosettle, stick, or clump to themselves or on the insides of the fluidpath elements. One approach to over come this is to start the flow witha higher or more rapid velocity than is used for the majority of theinjection. A second approach is that of FIG. 7E. The injectate is pulledback fluid path 1 while a purging or physiological solution is injectedat the same flow rate down fluid path 2. This reverse flow in fluid path1 will help loosen any clumping or adhesions, without pulling any fluidout of the patient. Then, when the injection starts to flow in theforward direction in fluid path 1, there is suction out fluid path 2until it removes a volume approximately equals the volume that had beenpreviously injected down fluid path 2. Similarly, this helps ensure thatlittle or none of the purge fluid is delivered to the tissue. Then theinjection can proceed according to the preferred delivery scheme.

FIG. 7F shows the fluid path elements of FIG. 7A, with an exemplaryfluid flow indicated. Fluid path 1 carries the therapeutic injectate,fluid path 2 is delivered simultaneously with a lower viscosity“lubricating” fluid. The two fluids flow together to the end Bd of fluidpath element B. At end Bd, the lubricating fluid is removed by suctionon fluid path 3 so that a more concentrated cell carrying fluid isdelivered to the tissue. From this example, it is apparent that all ofthe functions described above with respect to two fluid paths can berealized with the 3 fluid paths of FIGS. 7A and 7F.

In some embodiments, a solid needle, commonly called a stylet is insidethe hollow fluid path element when it is inserted into the tissue. Thisis often done so that a core of tissue does not fill the hollow fluidpath and to minimize the damage to the tissue. However, when the solidstylet is withdrawn, it created a suction on the tissue at the tip,and/or the hollow fluid path is filled with air. It is generallydesirable that this air not be injected into the patient, especiallywhen the delivery is through catheters in the blood vessels. A multiplefluid path embodiment similar to that of FIG. 100 b can be usedeliminate this problem. When fluid path element A is originally a solidstylet, fluid path 2 can be used to slightly pressurize the space aroundelement A, so that as it is moved, fluid flows to fill the space. Thisrequires a seal at the proximal end of fluid path element B. Such seals,often made of an elastomeric material, are well known in the medicalarts, especially in regards to catheters in interventional and specialprocedures labs. Hemostasis valves and needleless ports are examples ofsimilar devices.

It is preferred that the injection of marking or track filling fluid isautomatically coordinated with the withdrawal of the effector. Theposition of the effector can be tracked with various methods known inthe art. Similarly, the injection of the therapeutic fluid can besynchronized with the motion of the effector, so that the track left bythe effector is filled with therapeutic fluid. The marking could occursimultaneously with the indication to the user that the injection isover and that the needle can be removed, as was discussed above.

While stylized fluid path elements have generally been discussed andcan, for example, be concentric cylinders. In many cases this optimizesthe uniformity of flow and helps preserve laminar flow. Eccentriccylinders can generally be easier to manufacture, especially if theytouch and have a wall in common, and they may have some benefits in use.In addition to not demanding concentricity, most of the concepts of thisinvention can also be accomplished with parallel or adjacent fluid pathelements, or in fact totally separate fluid path elements that only meetor connect at the patient. In the connection, they may then have or nothave concentricity, dependent upon the need to be separate or mixed andupon the details of the fluids. The assembly of structures in suchembodiments use techniques well known in the medical device anddisposables art. Gluing can be used to assemble separate molded and/ormachined parts. Insert molding can be used advantageously in someinstances to capture metal or plastic elements in other plasticelements. Co-extrusion can create fluid path elements of significantlength. Assembly with elastomeric seals is applicable to someembodiments such as those of FIGS. 5C and 5D.

In addition, the drawings of FIGS. 7A to 7F are for clarity ofunderstanding and are not to scale in length, width or proportions. Thefluid path elements may change diameter, cross section, shape, or sizeor taper over their length. Example geometries are discussed in relationto the examples applications. Generally for clarity and consistency, thetherapeutic fluid is discussed as being delivered through fluid path 1,however, generally the fluids can be transmitted through any of thefluid paths provided that the fluid path elements are compatible withthe fluids and the shear stresses are sufficiently low. The walls offluid path elements are shown as lines. As discussed elsewhere,turbulence is generally damaging to cells.

The necessary rounding or tapering of any edges depends upon thethickness, roughness, and fluid flow parameters to be used in aparticular case. The generation of turbulence can be modeled and avoidedusing computational fluid dynamics packages as described elsewhere. Insome embodiments and applications the effector itself does not need topenetrate the tissue but is inserted through a needle, through apreviously made track, or over a guidewire. In others, where it needs tobe strong and sharp enough to penetrate the tissue, there will becompeting design needs on fluid path element wall thickness and edgegeometry. It is anticipated that because the therapeutic fluid isflowing into the tissue at that point and entering an uncontrolledgeometry, the desire for laminar flow can be relaxed and that the sharpedges will most likely be beveled in any event, which will minimize anystep transitions and their subsequent generation of eddies.

Pump/Injector System and Container

In several embodiments of the present invention, pump/injector system100 is designed to mechanically deliver fluid to tissue and,particularly, the myocardium. As discussed above, among the potentiallybeneficial fluids that can be delivered, autologous bone marrow-derivedprogenitor cells offer promise in the treatment of diseases of the hearttissue such as occurs in congestive heart failure and dopamine producingcells offer promise in treating, for example, Parkinson's disease. Inlight of these and other applications, in several embodiments of thepresent invention, pump/injector system 100 was designed with specialattention to, for example, the handing and delivery of such cells.Features preferably present in several embodiments of a pump or injectorfor delivery of such cells include: 1) consistent, repeatable dosagesize, 2) a 15-30 ml total volume, packaged in a disposable container,and/or 3) a specified volume to be mechanically injected on demand in adefined period of time, for example, one second or less. Thus, inseveral embodiments of the present invention, pump/injector system 100provides consistent and accurate delivery of a specified volume of fluidinto, for example, the myocardium of the heart or the brain, ensuringthat the total volume is accurately distributed across the total numberof injections and delivered at an appropriate rate.

In an embodiment illustrated in FIG. 8A, a disposable container orsyringe 50″ can be snapped securely and reliably into place withpump/injector system 100″ in a simple, two-step operation. The easy andsecure mounting of disposable container or syringe 50″ reduces operatoreffort and time while also reducing the risk of error. The simpleoperative attachment enables syringe plunger 56″ to be pushed forwardfor injections and withdrawn for removal and disposal (if necessary)with little user effort. As illustrated in FIG. 8A, injector 100″includes a seating or cradle section 105″ for receiving syringe 100″. Arearward section of syringe plunger 56″ includes an attachment flange58″, which cooperates with a retaining seating 112″ on a forward end ofinjector drive member 110″. Flange 58″ and/or seating 112″ can, forexample, be formed from one or more resilient materials (for example,polymeric materials as known in the polymer arts) so that a snap fit isformed to securely retain syringe 50″ within seating 105″ and withinoperative connection with injector 100″. To attach syringe 50″ toinjector 100″, syringe 50″ is angled with respect to injector 100″ asillustrated in the upper left portion of FIG. 8A. In this angledorientation, flange 58″ is first placed in connection with seating 112″and then syringe is moved into alignment with seating 105″ asillustrated in the upper right portion of FIG. 8A.

As illustrated, for example, in FIG. 8B, mechanical drive, drive memberor piston 110″ pushes the disposable syringe's plunger 56″ forward with,for example, a screw drive. Injector drive mechanisms are, for example,described in U.S. Pat. Nos. 4,677,980, 5,383,858, 6,585,700, PublishedPCT International Patent Application No. WO 02/04049 and U.S. patentapplication Ser. No. 10/921,083, filed Aug. 18, 2994, the disclosures ofwhich are incorporate herein by reference. The screw drive can, forexample, be powered by a highly accurate stepper motor 120″ and a small,powerful battery 122″. This reliable method of driving a small pumpmaintains accuracy and power in a suitably small package.

As illustrated, for example, in FIG. 8C, a custom assembly 1100including screw-drive components 1110 and portions of an electromagneticmotor 1120 can be inserted into a handle/housing 1130 containingadditional circuits and motor components as known in the art, thuscompleting the motor assembly necessary for driving an injection. Thissignificantly smaller approach to a mechanical drive assembly providesan injector suitable for even the smallest fluid injection volumes.

FIG. 8D illustrates use of an embodiment of a pump/injector system 1200of the present invention for the use of injection of, for example,SPHERAMINE into the brain of a patient. In this embodiment, the injectorsystem includes a syringe pump 1210 to deliver the SPHERAMINE from asyringe 1220 to the patient. As further described in connection withFIG. 9C below, syringe pump 1210 can be enclosed in a sterile bag orother containment system or barrier. In the illustrated embodiment,syringe 1220 is placed in fluid connection with a needle 1230 localizedby a stereotactic frame or similar localization device 1240 via a lengthof flexible tubing 1250. As known in the art, needle 1230 can beprovided with a removable stylet to prevent coring upon advancementwithin tissue. Further, needle 1230 can pass through a cannula inoperative connection with stereotactic frame 1240. As compared tocurrent manual techniques, connection of syringe pump system 1210 toneedle 1230 via flexible tubing 1250 isolates needle 1230 andstereotactic frame 1240 from force, torque, or vibration.

As compared to current manual injection of SPHERAMINE, pump drivensystem 1200 of the present invention can also provide the benefits offlow, volume and pressure control and auto loading. Pump system 1210 isalso capable of reversing before injecting, delivering the dose inpulses or conducting a two-phase or multi-phase injection.

In several embodiments, the injectate of interest (for example,SPHERAMINE) can be present only within needle 1230 and a flushing fluidis used to inject the SPHERAMINE into the brain of the patient. Suchembodiment can, for example, limit shear experienced by the injectate.

FIG. 8E illustrates another embodiment of an injection system 1300 ofthe present invention suitable, for example, to inject cells (forexample, SPHERAMINE). In this embodiment, a syringe pump or otherinjector 1310 in operative connection with a syringe 1320 including afluid therein is used to mechanically or hydraulically drive a syringe1330 (via tubing or conduit 1324) that delivers SPHERAMINE to thepatient via a needle 1340 in operative connection with a stereotacticframe 1350. Once again, syringe pump 1310 and/or other components ofsystem 1300 can be enclosed in a sterile bather. By using syringe pump1310 or other drive mechanism to drive syringe 1330 containing, forexample, SPHERAMINE, as opposed to delivering the SPHERAMINE directly asillustrated in FIG. 8D, the fluid path length for the SPHERAMINE isreduced.

In several embodiments, the pump/injector systems of the presentinvention can be programmed to deliver a calculated volume, whichequates to a predetermined amount of viable cells based on an algorithmsuch as a statistical algorithm. For a desired amount of stem cells thealgorithm determines the required volume for a given time in the lifecycle and processing time of the drug.

For example, it is known that FDG decays with a half-life of 110 minutesfrom the time it is fabricated. It is also known that living stem cellshave a nominal life under the conditions they are subjected to duringdelivery, and experience a settling or packing as a result of time andsyringe/vial orientation

Given this information, the injector calculates from the time the cellswere cultured to the present time to determine the percentage of live(viable) cells remaining in the syringe/container. If there is anysignificant settling that occurs over time, the injector can calculatethe amount of settling and deliver a flow profile that, for example,includes less volume in early injections and more volume in laterinjections, or vice versa to provide a consistent amount of viable cellsfrom the first to the last injection for a given container. Thealgorithm can calculate the volume required for each injection todeliver the predetermined amount of viable (viable cell count) cells foreach injection. Other factors such as a slide cell count or temperatureof the culture can also be considered in the algorithm. If cellmeasurements are taken periodically during a delivery session, this canbe used to update the algorithm.

Further, if stem cells are known to require a high flow rate to breakthem loose from, for example, the needle, tubing, or syringe, the flowcan be tailored to deliver a high flow at the beginning of the injectionto break the cells free and taper off to give a steady delivery of cellsover time.

User Interface System

The surgical field is often a crowded, stimulus-filled environment. Theuser of the devices and systems of the present invention is oftenwearing layers of surgical gloves, a gown, mask and face shield. Userinterface system 700 (see FIG. 4) of fluid delivery system 5 preferablyprovides easy, adequate and appropriate feedback and input control tothe user during operation.

The feedback or information provided to the user can include, but is notlimited to: total volume injected; volume remaining to be delivered;injection dosage volume; status of an injection in progress; map ofinjection area (for example, a 3-D computer generated map, position ofinjections made, position of injections to be made, cell viability,number of cells injected, number of cells reaming, and flow rate. Thecontrols provided to the user can include, but are not limited to:dosage volume; injection start/stop; injection position, and flow rate.The controls afforded the user further preferably provide the user readyaccess to, and accurate control of, a repeatable, accurate andconsistent dose size, without the inherent inaccuracies of a manuallycontrolled injection.

In one embodiment as illustrated in FIG. 9A, a switch assembly 1350 isoperatively connected in the vicinity of patient interface 400 (forexample, to a depth stop mechanism 1400—either permanently ortemporarily). Depth stop 1400 can, for example, include an abutmentsurface 1410 that abuts tissue and thereby limits the depth to whichneedle 400 penetrates tissue. The position of abutment face 1410relative to the distal end of needle 400, and thus the penetrationdepth, can, for example, be adjusted via threading 1420 or otheradjusting mechanism. Switch assembly 1350 can be used to triggerdiscrete injections. A button mechanism 1360 or other interface can, forexample, provide tactile feedback to the user. Also, a small LED 1370connected to the switch circuitry can be used to visually signal thestart and stop of each injection.

In the embodiment of FIG. 9B, the system subassembly of pump/injector100″ and syringe 50″, which is shown in operative connection with switchassembly 1350 and depth stop mechanism 1400, is attachable to and wornby the user. Part or all of control system 200 can also be containedwithin the injector housing. The user can, for example, operate thesystem by attaching the system subassembly to the forearm of the user,thereby freeing up both hands to manipulate the fingertip unit/switchassembly 1350 (see FIGS. 9B through 9D) in the surgical field. With bothhands free, the user has a greater degree of physical dexterity and anincreased control of accuracy and precision during the procedure.Moreover, a user wearable injector system can free valuable space in thesurgical field. FIG. 9C illustrates the use of a sterile barrier 1450 toenclose at least a portion of the fluid delivery system of the presentinvention. In the embodiment of FIG. 9D, bather 1450 is, for example, aflexible barrier (for example, a flexible polymeric material) that iswearable by the user of the system subassembly of pump/injector 100″ andsyringe 50″. The user can, for example, don the wearable systemsubassembly of pump/injector 100″ and syringe 50″ outside of the sterilefield and then don sterile barrier 1450, covering the subassembly, tomaintain sterility in the sterile field. Alternatively, the subassemblycan be provided in sterile condition (for example, in sterilepackaging). The pump/injector 100″ and container 50″ can be disposableafter a single use. Pump/injector 100″ can also be sterilizable toprovide for multiple uses. Sterile barriers can also be used inconnection with nonwearable embodiments of pump/injector 100″ and otherinjectors of the present invention.

The system subassembly of pump/injector 100 and syringe 50 and othersystem components can, for example, be made to be MR compatible for usein an MR environment as described, example, in U.S. Pat. Nos. 5,494,035,Published PCT International Patent Application Nos. WO 02/082113 and WO03/006101, and U.S. patent application Ser. No. 10/916,946, filed Aug.12, 2004, now U.S. Pat. No. 7,315,109 the disclosures of which areincorporated herein by reference, as well as in other imaging systemenvironments.

As illustrated in FIG. 9D, the system subassembly of pump/injector 100and syringe 50 can be attached to the user with an attachment mechanismsuch as a simple adjustable armband or other strapping 1460. Strapping1460 can, for example, include a hook-and-loop type fastener such asVELCRO® or other fastening mechanism as known in the fastening arts.Upon attachment of the subassembly to the forearm of the user, a set ofcontrols located on device 100″ preferably faces “upward” toward theuser's eyes while in use. The proper orientation of displays andnomenclature facilitates use by both right- and left-handed users. Witha clear view of the display on the forearm unit, the user has readyaccess to and knowledge of the variety of functional parameters of thedevice, reducing the possibility of error or miscalculation.

As illustrated in FIG. 9E, information displayed on one or more displaysof the forearm unit can, for example, be clearly readable from up to 30″and provides continuous indication/feedback to the user of, for example:injections remaining; injections made; dose volume; volume remaining;volume injected; injection in progress; injection complete; devicestatus, battery power injection etc.

Information/feedback to the user can alternatively or additionally beprovided using a display mounted to remain in the user's field ofvision. By placing pertinent information in the user's view at alltimes, the user may consult the information without taking the user'seyes off of the procedure at hand. In the embodiment of FIG. 9F, forexample, a display 1470 in communicative connection with injector system100″ is mounted on a frame or support 1480 attached to a headband 1490worn by the user.

In-Line Measurements/Sensor Feedback/System Control Architecture

A. Patient Physiological Parameters

In one embodiment of the present invention, patient interface 400includes or has in operational connection therewith one or morephysiological measurement devices, systems or function. For example,such devices can determine the location of damaged tissue, such asischemic and infarcted areas of heart tissue.

Biosense-Webster, a J&J/Cordis subsidiary, has, for example, developed asystem to create functional maps of cardiac electrical and mechanicalactivity using catheter-mounted electrodes. That NOGA catheter is usedin the cath lab to determine the location of ischemic and infractedareas of the endocardial wall. It is useful in assessment of treatment,since ischemia is caused by reduced oxygen delivery to cardiac muscle,and can be corrected by procedures that restore blood flow, whileinfarction is associated with unrecoverable dead tissue. Implantation ofcells would follow different strategies based on the diagnosis ofischemia versus infarct.

In the NOGA system, the location of the contacting electrodes is trackedin real-time by a standard electromagnetic tracking system. Data is usedby a computer to create maps of the cardiac activity. Data can besampled from inside the heart (see following) or from the outside usingcatheters or sensing needles.

U.S. Pat. No. 6,892,091, the disclosure of which is incorporated hereinby reference, discloses a catheter capable of mapping the electrical andmechanical activity of the heart by sampling the voltage and mechanicalstrain at unique points on the endocardium. A three-dimensionalcolor-map of the activity is created by associating data with a locationof the sampled tissue determined by electromagnetic tracking of thecatheter tip.

Another way of monitoring ischemia or hypoxia in cardiac tissue isthrough the use of catheter-mounted or needle-mounted oxygen probes.These devices are electrochemical devices mounted in or upon invasivedevices that make contact with tissue. These devices are capable ofresponding to the partial pressure of oxygen present in and aroundperfused tissues. Several commercial devices are available fromOxford-Optronix of the Oxford, United Kingdom

In several embodiments of the present invention a therapeutic device iscoupled with a diagnostic device to inject therapeutic fluids, cells,cell carriers (including, for example, beads), for example into sites ofdamaged heart tissue. As illustrated, for example, in FIG. 3, system 5can, for example, include: (1) patient interface 400 (e.g. catheter orneedle) to inject therapeutic fluids, emulsions, suspensions, gels,solids etc. into tissue as described above; (2) one or more sensors,measurement devices or monitors 600 (for example, mounted in or upon thepatient interface 400 or otherwise placed in operative connection withthe patient) to measure one or more biophysical properties of thepatient and/or the patient tissue; (3) one or more imaging systems 500to display regions of the patient (for example, ischemia or infarctiondistinguishable from healthy tissue); and (4) a feedback system by whichan operator can use imaging system 500 to guide patient interface 400(for example, with sensor(s) as described above) to a region of damagedtissue (for example, ischemic tissue) to inject therapeutic fluids,cells, or cell carriers

The sensing device(s) can, for example, make direct contact with thetissue to distinguish among well-perfused, or infarcted (dead) orischemic (stunned) tissue, presuming that injection into ischemic tissueis more likely to restore function to the affected area. Based on themeasurement, system 5, through control system 200, can allow or disallowthe injection. Preferably, system 5 at least alerts or informs theoperator of the tissue condition prior to delivery of a therapeuticfluid.

System 5 can also include a measuring or sensing device to detect theamount of blood flow or capillary perfusion in tissue. In oneembodiment, the sensor makes direct contact with the tissue and respondsrapidly to change in blood volume in a perfused tissue. One example ofsuch a device is a thermistor, which is sensitive to rapid changes inblood volume as indicated by temperature change at the contact point.The thermistor changes its electrical conductivity in response to smalltemperature differences. Sensitivity of the measurement can be increasedby using a pair of thermistors with one serving as a reference.

In another embodiment, the measuring or sensing device is a contactingor a non-contacting infrared light source and an infrared sensorarranged as a pair. This sensor pair responds to small changes in bloodperfusion by sensing reflected and scattered light in tissue. Highlyperfused tissues are easily distinguished from ischemic or infarctedtissues because of the optical properties of blood with respect to thescattering and absorption of infrared light. This principle is known inthe art (see, for example, U.S. Pat. No. 6,122,536, the disclosure ofwhich is incorporate herein by reference), but sensing systems thatprobe perfusion of tissue on percutaneous medical devices are unique.

To position the sensor residing near the distal tip of patient interface400 (for example, a catheter, needle, or endoscope), an additionalminiaturized device can be provided to determine the sensor locationwith respect to the tissue under treatment. The location of the sensorcan then be superimposed upon the image of the tissue displayed for theoperator by imaging system 500. A medical positioning system of thistype is described, for example, in U.S. Pat. No. 5,526,812, thedisclosure of which is incorporated herein by reference. That systemuses an electromagnetic field and multiple antenna loops to sense thefield and to triangulate sensor position for processing by a computergraphics system. As described above, the present invention can provide amap of, for example, blood perfusion in tissue in near real-time priorto the administration of therapy.

Physiological parameters such as respiration and/or heart function canalso be measure to, for example, provide a positioning function, agating function or an injection timing function. For example, FIG. 17Aillustrates the use of an electrocardiogram (EKG) which can be used tomeasures heart movement and synchronize injection. In this system,control system 200 is in operative connection with the EKG monitor (forexample, part of monitor system 600), which measures the heart'sactivity. Control system 200 uses that information to control pumpsystem 100 and/or patient interface 400 to, for example, deliver fluidwhen the heart muscle is relaxed (during diastole), enabling greaterfluid delivery and distribution.

Marking and Mapping During Delivery

In several embodiments of system 5 marking of delivered injectate andmapping of tissue regions is provided. During the injection procedure,one goal of marking is to enable the doctor or operator to determinewhat tissue has been treated, both to avoid double treatment and toensure sufficient coverage of the area to be treated. Marking also helpsprovide uniformity of treatment over the tissue surface, with the optionof quantifying the treatment in two or three dimensions. These resultsare especially useful in external heart treatments and dermatologicaltreatments. The marking can be such that it is used long term to monitortissue response or cell migration. An ancillary benefit is that some ofthe marking mechanism can optionally help keep the injectate in thetissue.

One set of marking embodiments marks the surface of the tissue beingtreated, to indicate the location of the needle puncture or anapproximation to the spread of the injectate within the tissue as orafter the injection occurs. These markings may be visible to the eye,(either unaided and aided) such as dyes applied by a “rubber stamp” typeapplicator of U.S. Pat. No. 5,997,509, the disclosure of which isincorporate herein by reference. U.S. Pat. No. 6,322,536, the disclosureof which is incorporate herein by reference, discloses sutures or othersurface mechanical devices. Embodiments also include the deposition ofpowders or foams through, for example, a second delivery channel asdescribed above. Biodegradable solid segments can also be beneficiallyused as markers, with the added benefit of sealing the wound. Adhesivedots of tissue scaffolding material are one option. Gels or solid barbedpins, optionally filling the needle tract to reduce back flow, areanother option. The applicator or patient interface itself can create amechanical mark. It can, for example, use vacuum to hold the tissuebeing injected, thereby raising a small bleb. The hole made during theinjection can bleed, and the bleeding or clotting can act as a mark.Alternatively or additionally, the process of touching the tissuesurface can roughen the surface, providing visual indication. Further, asmall region of tissue can be cauterized, possibly cauterizing theinjection site itself, providing both marking and sealing the tissue toreduce injectate leakage. Devices for augmenting the operator's visioninclude endoscopes or thorascopes, microscopes, and cameras which can besensitive to visible or non-visible electromagnetic radiation.Fluorescence can also be used beneficially, where the output of themarker is in the visible range as it is excited by a possibly moreintense light at a higher invisible wavelength.

Another marking approach is to mark the injectate itself. This has thebenefit of allowing 3D visualization of the injection if an imagingsystem of some type is used. An injectate rich in water can, forexample, be differentiated from many tissues using MR imaging. Additionof imaging contrasts—for example, ultrasound, X-ray/CT, or MR contrastto the injectate—can improve visualization by the respective imagingmodalities. A radioactive component or PET tracer could be added to theinjectate or to cell surface for imaging via nuclear medicine. PublishedU.S. Patent Application No. 2003/219385A1 and Published PCTInternational Patent Application No. WO 2005/072780A2, the disclosuresof which are incorporated herein by reference, disclose two methods formarking cells so that it is possible to monitor cell proliferationand/or migration after the delivery as well as the delivery processitself. Alternatively, the cells being marked can be non-active cells sothat their only use is to transport the marker. The marker could be inseparate particles that could be solid, liquid, for example in liposomesor solid shells, or gaseous particles such that they are visible underone or more medical imaging modalities.

The marking process can involve a reaction during the injection. Forexample, injecting an alginate and the calcium salt solution requiredfor polymerize enables a liquid to be delivered and a solid to be formedin the tissue. An alternative is to have the reaction be between themarking device and the tissue. An example is a marker that changes coloror imager contrast properties upon exposure to air, water, or a specificpH, such as present in commonly available pH indicators. Alternatively,the injectate can cause a quick physiological response, similar to amosquito bite, with the resulting bump indicating the injection site.

If a computer based system is used to visualize or augment thevisualization, then one of several virtual marking systems can be used.One embodiment of such a device or system incorporates anelectromagnetic field position measuring system. Commercial or researchsystems are available from a number of manufacturers (EndocardialSolutions (EnSite 3000), Biosense Webster/J&J (CARTO XP, NAVI-STARcatheter), Medtronic (LocaLisa), Boston Scientific (RPM RealtimePosition Management System)). By measuring the 3D position of theinjection effector when an injection is given, a 3D model can be builtand displayed to the operator.

In another embodiment, a virtual marking system can be used if, forexample, an endoscope or thorascope with a camera is used. Using scenerecognition algorithms similar to those used to place the virtual firstdown lines on the football field, every time an injection occurs, theimaging system can mark or color that segment of tissue, providing avirtual ink mark on the surface.

As an alternative to tracking the actual injection sites, the markingsystem can lay down a grid or pattern that the user is to follow. In oneembodiment this is a physical grid, such as might be applied with ink ora label. Alternatively, the markings can be “painted” or drawn in realtime onto the tissue, for example with light or laser. The markings canbe static, or dynamic, for example moving or changing as the userperforms an injection to indicate where the next injection should takeplace. A similar guidance capability can be achieved virtually using acomputer and an image display mentioned above.

The computer guidance systems described above can be connected to arobotic system (for example, including patient interface positioningsystem 460) to automate the delivery. Such automation may be ofparticularly value when a very large number of injections are required(as in certain dermal implanting procedure as discussed above).

For many advantages, the marking is considered only during the deliveryprocess. However, there can be a benefit to verifying injectate deliveryin the time frame of hours, days, weeks or months. The more permanentmarking schemes described, such as solid particles or solid surfaces,can provide verification of delivery at any time. Biodegradable markerscan be used to provide marking for a desired time, and then degrade toreduce or eliminate any deleterious biological effects.

For those markers that are not part of the injectate, there can be asecond pump and fluid path to deliver the marker to the tissue surfaceor the tissue depth. The marker and therapeutic injectate can bedelivered through the same needle, with the marker going before,simultaneously, or after the therapeutic injectate. An example of thelater is a polymerizing marker that also acts as a plug to reduceleakage through the injection site. A fluid marker can be deliveredthrough a second independent fluid path, either to the surface or intothe tissue. For delivery into the tissue, delivery can, for example, bethrough a needle, a high pressure jet, a cutting edge, or a roughingsurface

A mechanical marker can, for example, be mounted on a depth stop asdescribed above or mechanically associated with an injectate effector asdescribed above.

Information Encoding

Maintaining traceability of cells and ensuring that they are deliveredfor their intended purpose is one of the challenges facing cell therapyproviders. In several embodiment of the present invention, cellcontainer 50, the injection fluid and/or the cells are encoded withinformation such as batch, date of manufacture, processing and/orharvest, and target patient. System 5 preferably includes a sensor orreader that is capable of reading the encoded information. Encoding ofsyringes/container and sensors used to read such information are, forexample, discussed in U.S. Pat. Nos. 5,383,858, 6,652,489 and 6,958,053and PCT Published International Patent Application Nos. WO 99/65548, WO02/056934, and WO 02/081011, the disclosures of which are incorporateherein by reference. The injection fluid can, for example, be encoded byproviding a detectable and distinguishable characteristic (for example,color). One or more physical and/or chemical identifying markers or tagscan also be added physically or chemically attached to the injectionfluid molecules or to the cells themselves.

In one embodiment, patient information can be entered into controlsystem 200 before cell-containing container or cartridge 50 is inserted.Upon insertion of container 50, the control system 200 (including, forexample, a sensor or reader on pump/injector system 100) “reads” theencoded label and verifies that the patient information on container 50matches the input information.

The above methodology can, for example, be particularly helpful inprocedures involving autologous stem cells or cells that have beenremoved from a patient, processed, and then implanted as an assurancethat the patient's own cells, and not another patient's calls, are beinginjected.

Encoding of cell container 50 can also include information about how thecells should be handled and maintained by system 5 such as mixing speed,temperature, and or maximum injection speed. Cell therapies requiringbuffers and other solutions to be mixed with them can provide thisinformation to the fluid handling system through encoding.

Another area in which encoding is useful is ensuring traceability of thetherapy itself. Pharmaceutical companies preferably take steps to ensurethat the therapy is used in the intended way and not misapplied.Verification of this use can be important. Encoded information can, forexample, be returned to the manufacturer as verification of proper use.

Processing of Cells Prior to Delivery

Cells can, for example, be stored and/or delivered in a transport orhibernation buffer solution as known in the art. An example of cellsdelivered in a hibernation fluid is SPHERAMINE. As described above,SPHERAMINE is formed of dopamine-producing human retinal pigmentepithelial (RPE) cells adhered to spherical microscopic carriers.SPHERAMINE can, for example, be implanted into the regions of the brainthat lack dopamine, where it produces dopamine in place of the patient'sown neurons which can no longer perform this function.

In current procedures for the injection of SPHERAMINE, approximately 1ml of RPE cells on the spherical microcarrier is, for example,transported and delivered with 3 ml of a hibernation or transport buffersolution in a 5 ml CYROVIAL®. In general, Cryovials are tubularcontainers or vials designed for storing and/or preservation biologicalmaterials (for example, at low temperatures) and are available from manysuppliers including Simport Plasiques LTEE Corporation of Quebec,Canada. Before injection of SPHERAMINE, the hibernation solution isremoved and the cells are washed to remove remaining hibernationsolution. In a typical procedure, the SPHERAMINE is allowed to settle tothe bottom of the cryovial. Using, for example, a syringe, the transportsolution is withdrawn by drawing down the fluid level to approximatelythe level of the SPHERAMINE (that is, to the 1 ml level).

A buffer solution such as Hank's Balanced Saline Solution (HBSS) is thenmixed with the SPHERAMINE to dilute the remaining hibernation solution.In that regard, after the SPHERAMINE is allowed to settle to the bottomof the vial, the HBSS is drawn down to the level of the SPHERAMINE.

Current equipment and manual techniques used in cell preparation (forexample, washing, buffer replacement and/or other fluid treatments) can,for example, lead to cell wastage and/or contamination. FIGS. 10A and10B, for example, illustrate two embodiments of containers or vials thatcan facilitate the preparation of cells for delivery. In each of theseembodiments, two tubes extend to different depths into the vialcontaining the cell slurry. In the embodiment of FIG. 10A, tubes 1502and 1504 are formed as part of the vial 1500. In the embodiment of FIG.10B, tubes 1512 and 1514 are built into or placed in operativeconnection with a cap 1516 that covers vial 1510. The tube (or otherport) that extends or is positioned to a higher level within the vial(tubes 1504 and 1514 on the right side of each of vials 1500 and 1510,respectively in FIGS. 10A and 10B) is operable to remove the liquidwithout disturbing the cell slurry that has been allowed to settle tothe bottom of the vials. The tube (or other port) that extends or ispositioned to a lower level (tubes 1502 and 1512 on the left side ofeach of vials 1500 and 1510, respectively in FIGS. 10A and 10B) isoperable to introduce a buffer or washing solution such as HBSS and toremove cells for injection into a patient.

During use, transport or hibernation fluid can first be withdrawn fromthe higher level tube without disturbing the settled cells. HBSS is thenintroduced through the lower level tube to wash through the cells. Aftersettling, waste fluid is then withdrawn through the higher level tube.Then, cells are withdrawn via the lower level tube for injection intothe patient. Among other benefits, the devices of FIGS. 10A and 10Beliminate the need to position a syringe just above the cell slurryduring the washing step, reducing the amount of operator skill and timeit takes to perform the washing step. This technique is also moreadaptable to closed operation than is manual pipetting, as is discussedelsewhere herein.

FIGS. 10C and 10D illustrate other embodiments of containers or vialsthat can facilitate washing of cells. FIG. 10C illustrates a cryovial1520 that is divided at generally the center thereof into a left side orregion 1522 and right side or region 1524 over a portion of the lengthof cryovial 1520 by a divider 1526 extending downward (in theorientation of FIG. 10C) from the top of vial 1520. A filter or filters1528 can be placed either at position A or position B (see FIG. 10C) onright side 1524 of the cryovial. Filter(s) 1528 is/are adapted to allowliquid to pass therethrough but to prevent passage of, for example,SPHERAMINE therethrough. Such a device can, for example, be used withany cells or other structures having a minimum size large enough to beexcluded from passage through filter(s) 1528. Flow of HBSS into cryovial1520 is represented by arrow A1, whereas flow of waste liquid out ofcryovial 1520 is represented by arrow A2. As represented by arrow A3,cells (for example, SPHERAMINE) can be extracted from unfiltered leftside 1522 of cryovial 1520 or flushed out of cryovial 1520 byintroducing HBSS (or other fluid) into right side 1524 of the cryovial1520. As illustrated in FIG. 10D, the bottom of cryovial 1520 can berounded to facilitate flow through cryovial 1520. The embodiments ofFIGS. 10C and 10D, consolidate tubing by using cryovial 1520 as the washcontainer.

The embodiments described above and other embodiments of storage,transport and/or washing devices of the present invention can be usedwith systems that include a syringe and/or other pumps to, for example,add HBSS, withdraw waste materials, withdraw cell etc. One embodiment ofsuch a system is illustrated in FIG. 10E. System 1540 of FIG. 18Eincludes a closable cabinet 1542 or other enclosure that accepts, forexample, a modified cryovial 1520 as described above, as described inconnection with FIG. 10F below, or another container. In the illustratedembodiment, syringes 1544 and 1546 are used to effect flow within andout of system 1540. A sample port 1548 is provided for a fluid sample toexit the system. System 1540 can further include fluid path connectorsto connect to ports/tubes on the top of cryovial 1520 or needles topierce a septum on the top of, for example, vial 1560 of FIG. 10F. Acontrol system 1550 can be provided that can display information relatedto the status of the system via, for example, display 1552. In thatregard, FIG. 10F illustrates an embodiment of cryovial 1560 having apierceable septum 1562 suitable for use with an embodiment of a systemof FIG. 10E including one or more piercing needles 1554 and 1556 (in theillustrated embodiment).

In general, the vial remains in cabinet 1542 of system 1540 while fluidsare circulated and withdrawn from the vial for wash cycles, dilution, orsampling for an assay test. In that regard, one or more assay testingdevices 1556 (see FIG. 10E) can also be contained in the cabinet orotherwise be placed in operative connection with system 1540 (forexample, using cell or particle counting technologies, such as a coultercounter or an optical counter). In this embodiment, syringes 1544 and1546 can be large enough to contain all the initial wash solution andall the withdrawn waste solution. One or more valves can also beprovided to assist in controlling flow as known in the art.

System 1540 can be programmable, and can also include timers forindicating the status of various processes such as a wash/dilution cycleand/or to indicate when the vial is ready for removal. Moreover, system1540 can also be used as an “auto-loading” device to, for example,automatically fill a cartridge or syringe. The cartridge or syringe canthen be placed into operative connection with an injector or other fluiddelivery system for administration of the cell therapy to the patient.In one embodiment, system 1540 keeps track of vial contents, processing,etc. System 1540 can also create encoding devices including, but notlimited to, labels, barcodes, or RFID tags, for the vial to assist withtracking or indication of viability, concentration, time of preparation,and/or other information related to the status or condition of thecontents of the vial or prepared dose.

FIG. 10F illustrates two needles of different length piercing pierceableseptum 1562 of vial 1560. As described above, for example, in connectionwith FIGS. 10A and 10B needles 1554 and 1556 can be of different lengthsso that the shorter needle 1554 can, for example, be used to removehibernation solution or media and/or waste, while longer needle 1556can, for example, be used to introduce HBSS or other fluid to effectwashing or dilution and/or to remove cells.

In several other embodiments of the present invention, systems areprovided in which, for example, washing and dilution can be effectedusing a single device. Such devices can also form a part of the celltherapy delivery device or the entirety thereof. For example, FIG. 11Aillustrates a syringe 1600 including a syringe barrel having a plunger1602 slidably movable therein to draw fluid into the syringe barrel. Inthis embodiment, a first port 1604 is provided (for example, on the sideof the syringe barrel) through which a fluid such as HBSS can flow intosyringe 1600. A second port 1606 is provided (for example, on the sideof the barrel) through which waste can be removed from syringe 1600. Afilter 1608 is placed in operative connection with second port 1606 sothat cells and/or other materials to be injected are prevented fromexiting syringe 1600 through second port 1606. Cells can be injected viaan outlet port 1610 or syringe tip on the forward end of syringe 1600.Valves (not shown) as known in the art can be provided in connectionwith one or more of the ports of syringe 1600 to control flow.

FIG. 11B illustrates another embodiment of a syringe system of thepresent invention. In this embodiment, syringe 1620 includes a syringebarrel having a plunger 1622 slidably disposed therein. A generallyconical shaped transition region 1624 on a forward end of the syringebarrel connects to a neck 1625 which includes an outlet port 1626 at theforward end thereof. A second port 1628 is provided (for example, on theside of the neck). Second port 1628 or side port can be formedintegrally or monolithically with syringe 1620. Alternatively, a fluidpath section including an injection outlet port and a second port or canbe added via an attachment to a standard syringe in forming the systemof FIG. 10B.

The syringe of FIG. 11B can, for example, make use of gravity toseparate cells from liquid. When expelling waste via side port 1628 insyringe neck 1625, syringe 1620 can be oriented upward as shown in thebottom left of FIG. 11B. In this orientation, the cells settle to therearward portion of the syringe barrel. The liquid is ejected from thetop as plunger 1622 is slowly advance forward (toward syringe outletport 1626). When washing cells with, for example, HBSS syringe 1629 isoriented as shown on the bottom right of FIG. 11B. In this orientation,the cells settle to the forward portion of syringe 1620. HBSS or otherfluid can then be drawn into syringe 1620 by drawing plunger 1622rearward (away from syringe outlet 1626). Drawing the liquid intosyringe 1620 in this manner causes the liquid to flow through the cells(for example, SPHERAMINE). Appropriate valves as known in the art can beplaced in fluid connection with syringe outlet port 1626 and second orside port 1628 to control flow therethrough. The syringe system of FIG.11B isolates the cells within syringe 1620, reducing the possibility ofcontamination. Additionally, syringe 1620 can be a part of or can formthe entirety of a delivery device as, for example, described above.

FIGS. 11C through 11E illustrate an embodiment of a syringe 1650 of thepresent invention including a plunger 1652 having a volume or chamber1654 formed therein or in fluid connection therewith. Plunger 1652interfaces with the fluid within syringe barrel via a filter 1656, whichcan be considered as partially replacing the rubber cover commonly usedon the forward end of conventional plungers. Holes, pores or othertransport paths of filter 1656 are sized such that liquid may passthough, yet cells (for example, SPHERAMINE) cannot. Plunger chamber 1656has in fluid connection therewith an inlet 1658, including an inletvalve 1660, and an outlet 1662, including an outlet valve 1664 asillustrated in FIGS. 11C through 11E. Valves 1660 and 1664 can, forexample, include one-way check valves.

In a representative example of use of syringe 1650 of FIGS. 11C through11E, syringe outlet 1666 on the forward end thereof is first capped witha cap 1668 and plunger 1652 is drawn back, introducing HBSS into theplunger chamber 1654 (via inlet 1658) and through filter 1656 to contactcells held within the syringe barrel forward of filter 1656 (see, FIG.11C). In a second step illustrated in FIG. 11D, plunger 1652 is pushedforward, forcing waste to exit outlet valve 1664 in fluid connectionwith plunger chamber 1654. The above steps can be repeated to thoroughlywash the cells. In a subsequent step illustrated in FIG. 11E, cap 1668is removed from syringe outlet 1666 and outlet 1662 in fluid connectionwith plunger chamber 1654 is plugged or capped with a plug 1668. Uponforward advancement of plunger 1652, the cells are expelled from syringeoutlet 1666.

The syringe of FIGS. 11C) through 11E (as well as those of FIGS. 11A and11B) can include a sterile bag to contain the syringe. As with thesyringes of FIGS. 11A and 11B, syringe 1650 of FIGS. 11C through 11Eisolates the cells in the syringe, reducing the possibility ofcontamination. Also, syringe 1650 can form part of or the entirety of acell delivery system.

FIG. 11G illustrates another embodiment of a device or system 1680 foruse in connection with a cell therapy such a SPHERAMINE to provide aclosed system to wash and to deliver cells. Device 1680 can be used withany structure to be injected, including cell structures, that can besize excluded via, for example, a micropore filter.

Device 1680 includes a cell chamber or transport vial 1682 that includesa mechanism or fluid path to flush buffer solution (for example, HBSS)through chamber 1682 while retaining the cells in chamber 1682. In theembodiment illustrated in FIG. 11F, cell chamber 1682 includes an inletport 1684 through which a flushing buffer can be introduced into cellchamber 1682. A one-way check valve 1686 can be placed in fluidconnection with inlet port 1684. A septum 1688 can cover a first end ofcell chamber 1682. A filter 1690 covers the second end of the cellchamber 1682. Cell chamber 1682 is insertable within a housing section1692. An annular sealing member 1694 on an outer wall of cell chamber1682 forms a seal with an inner wall of housing section 1692. A bufferflush solution such as HBSS can be introduced through inlet port 1684 ofcell chamber 1682 to remove the hibernation or transport buffer solutionby moving. Waste flows out of device 1680 via an effluent port 1694 ofhousing 1692. A one-way check valve 1696 can be placed in fluidconnection with effluent port 1694. Device 1680 can also be used as theadministration syringe for delivery of cells by, for example, insertinga needle (not shown in FIG. 11F) into fluid connection with cell chamber1682 (for example, through septum 1688, through filter 1690 or throughanother port). A needle can also be placed in fluid connection witheffluent port 1694 of housing 1692. Filter 1690 can be moved to the backof chamber 1682 to facilitate use of device 1680 as a hand syringe byattaching a needle to the front of device 1680. Device 1680 isrelatively simple to use regardless of the skill set of the operator.

FIGS. 11G and 11H illustrates other embodiments of devices including acell chamber or transport vial that includes a mechanism or fluid pathto flush buffer solution (for example, HBSS) through the chamber whileretaining the cells in the chamber. These embodiments are somewhatsimilar in operation to those of FIGS. 11C through 11F. In the system1700 illustrated in FIGS. 11G and 11H, a cell chamber 1702 includes aninlet port 1704 through which a flushing buffer can be introduced intocell chamber 1702. A one-way check valve 1706 can be placed in fluidconnection with inlet port 1704. Cell chamber 1702 also includes aneffluent port 1708. A one way check valve 1710 can be placed in fluidconnection with effluent port 1708. As compared to the embodiment ofFIG. 11F, by moving the flow into and out of cell chamber 1702 (viainlet port 1704 and effluent port 1710 during, for example, repeatedreciprocation of plunger cell chamber 1702 relative to housing 1714) tothe same side of a filter 1712 (with the cells isolated on the otherside of filter 1712) packing of cells on filter 1712 is prevented. Inthat regard, filter 1712 is washed of cells each time buffer solution isintroduced. A sealing member 1703 can be placed in connection with anouter wall of cell chamber 1702 to form a seal with an inner wall ofhousing 1714.

In the embodiments of FIGS. 11G and 11H, a needle or other deliverydevice is attachable to, for example, a connector 1716 (for example, aluer type connector or other connector as described herein) on the endof housing 1714 (for example, a graduated cylindrical housing) withoutthe requirement of puncturing a septum.

In FIG. 11H, plunger/cell chamber 1702 is positioned within housing1714. A cap 1718 is provided for use in connection with connector 1716.Further, cap 1720 is provided for use in connection with effluent port1708 during an injection using device 1700.

With cap 1718 in closing connection with connector 1716 and cap 1720 notin connection with effluent port 1710, rearward movement of plunger/cellchamber 1702 relative to housing 1714 (that is, movement of plunger tothe right in the orientation of FIG. 11H), results in drawing fluidthrough inlet port into device 1700. Forward movement of plunger/cellchamber 1702 relative to housing 1714 result in effluent exitingeffluent port 1710. Repeated reciprocation of plunger/cell chamber 1702result is fluid treatment (for example, washing and/or bufferreplacement) of the material (for example, cells) within device 1700. Toinject using device 1700, cap 1720 is place in closing connection witheffluent port 1710 and cap 1718 is removed form connection withconnector 1716. A needle can, for example, be placed in fluid connectionwith connector 1716.

Device 1700 can be used to process any type of solution have solidssuspended therein. Filter 1712 is used to separate such solids via sizeexclusion.

Moreover, as illustrated in the embodiment of FIG. 11I, a plunger 1750can be used in connection with a conventional cryovial or othercontainer 1760 to effect fluid treatment (for example, washing and/orbuffer replacement) within a standard or convention cryovial or othercontainer 1760. In general, plunger 1750 includes a filter 1754 on thedistal end thereof to prevent cells (and/or other material in vial 1760)from entering either of two fluid pathways within plunger 1750. Fluid,which can pass through filter 1754, can enter vial 1760 through thefirst pathway and can exit vial 1760 through the second pathway formedthrough plunger 1750. One way check valves as described above can beused in connection with the first and second fluid pathways. A vial cap1762 is first removed from vial 1760 (via, for example, threading 1764)as known in the art. Plunger 1750 is then placed within vial 1760.

In the embodiment illustrated on the left side of FIG. 11I, the firstfluid pathway (which can, for example, be in fluid connection with abuffer solution) is a conduit 1756. A first check valve 1757 can beplaced in fluid connection with conduit 1756 so that fluid can flow intovial 1760 through conduit 1756 but cannot exit via conduit 1756. Thevolume around conduit 1756 within plunger 1750 provides a second fluidflow pathway 1758 for effluent fluid to exit through plunger 1750 towaste. A second check valve (not shown) can be placed in fluidconnection with second fluid flow pathway 1758. A sealing member 1759(for example, an elastomeric, O-ring) is provided on plunger 1750 toform a seal with an inner wall of vial 1760. Upward (in the orientationof FIG. 11I) motion of plunger 1750 relative to vial 1760 results indrawing of fluid into the system. Downward motion of plunger 1750results in forcing effluent out of the system.

In the embodiment of plunger 1750 a on the right side of FIG. 11I, firstfluid pathway 1756 a and second fluid flow pathway 1758 a are created bya divider 1753 a in plunger 1750 a. A first check valve 1757 a is placedin fluid connection with first pathway 1756 a and a second check valve1757 a′ is placed in fluid connection with second pathway 1756 a. Otherlike elements are numbered similarly to the numbering of correspondingelements of plunger 1750.

Further embodiments of cell processing and/or delivery systems of thepresent invention are illustrated in FIGS. 12 through 13D. The cell washsystem of FIG. 12 washes the cells using multiple dilutions and gravitysedimentation to remove transport or hibernation solution/medium fromthe cells so that they can be injected. The separation time can bereduced by employing centrifuging, as is known to those skilled in theart, at the expense of greater forces on the cells. The illustratedembodiment uses gravity.

The fluid path is closed and presterilized so that there is minimalchance of contamination of the cells. In addition, the closed systemallows the preparation of the cells to take place in, for example, acath lab, a neurosurgery suite, or any other place, preferably near thedelivery procedure, without the need for a sterile hood.

With reference to the fluid path schematic illustrated in FIG. 12, thereare three main fluid containers: a wash solution container 2010, hereshown for example as an unvented collapsible bag, a cell container 2013,here shown as for example a 5 ml cryovial, and a waste container 2011,also shown for example as a collapsible bag, either unvented or vented.There are various fluid path elements connecting these containers. Thereare three pumps, shown here for example as syringe pumps, 2020, 2021,and 2022. The syringes and fluid path should be formed of materials thatare compatible with the cells, for example polypropylene, polyethylene,and Teflon are suitable. In some situation it is desirable to have nosilicon lubricant. Suitable syringes are manufactured for example byHenke Sass Wolf GMBH (HSW) of Tuttlingen, Germany. The wash solutioncontainer comes prefilled with the preferred wash solution for thespecific cells, for example normal saline, Hanks Balanced Salt Solution(HBSS), or any other suitable liquid. The remainder of the fluid pathcan also be filled with wash solution, or it may be filled with air. Ifany of the fluid path elements contain air, then it will be necessary tofill them with liquid before starting the wash operation. (Note that itis normal and even desirable for some air to remain in the cell storagevial assembly 2013 a). Connectors as described herein can be used toconnect various fluid path elements.

The system of FIG. 12 includes a cap 2014 a that is constructed tocooperate with the cell storage vial 2015 a. The cap 2014 a has threepenetrations or passages passing therethrough. The first penetration hasa tube 2016 that goes down to close proximity or touching the bottom ofthe vial 2015 a. This is the inlet for the wash solution and it will beused at the end of the process to remove the washed cells as describedabove. The second penetration has a tube 2017 does not go as far down asthe first tube 2016. Tube 2017 is used to remove the mixture of the washsolution and the contents of the vial. As described above, it does notgo as deep as tube 2016 so that it does not draw cells therein whenremoving the mixed fluid. The position determines the amount of fluidcontaining cells that remains after the fluid has been removed. In thisembodiment, it is approximately 1 ml. The third penetration is an airpath 2018 with a sterile filter so that air can move in and out as thefluid level changes in vial assembly 2013 a, without compromisingsterility.

To wash cells, vial 2015 a is removed from the system. The lid 2014 b isremoved from the vial assembly 2013 b containing the cells, and vial2015 b is screwed onto lid 2014 a. The system is only open to possibleairborne contamination for the few seconds it takes the operator to makethis switch. If need be, this could be done in a sterile hood, glovebox, or bag. Moreover, the transport cryovial can include ports thatcooperate with a cap of the present system to eliminate the need to openthe vial. However, it is anticipated that the atmosphere of the surgicalor special procedures suite will be sufficiently clean that this briefexposure entails an insignificant risk of contamination.

Once the cells are in place in the wash system, after sufficient time toallow for settling of the cells, for example 3-7 minutes, syringe pump2020 is activated and the excess hibernation or transport solution isdrawn from vial 2015 b into syringe 2030 through a one way valve. Whensyringe 2030 is moved forward or upward, fluid is driven out the one wayvalve into waste bag 2011 or sampling syringe 2033, depending uponposition of stopcock 2041. The sampling syringe can be used to samplethe fluid that was just withdrawn to check for bacterial contaminationof the cells, to verify that the wash is proceeding satisfactorily, orthat it has been sufficient.

To avoid bubbles in the fluid path, it is preferable that the fluidlevel in vial 2015 b never get so low that tube 2017 draws in air. Asensor, not shown, could be incorporated to measure fluid level in vial2015 b. However, if air is drawn through tube 2017, is not a significantproblem because the bubbles will simply move into the waste container2011 or sampling syringe 2033. In an alternative embodiment, one simplydoes this every time, pull out excess fluid so that air is pulled intotube 2017, to ensure that the fluid level returns to a known positionwithout any need for sensors.

To deliver wash solution from container 2010 to vial 2015 b, an optionalstopcock 2042 (or other valve) is rotated so that fluid can be pulledfrom the wash solution container 2010. Stopcock 2040 is rotated intoposition so that fluid path 2050 is disconnected and fluid can flow from2049 to 2016. Wash solution is pulled from the wash solution container2010 through one way valve 2046 by pump 2021 pulling down on the syringe2031 plunger. After pulling in sufficient fluid for the wash, syringepump 2021 pushes the plunger upward and fluid flows out one way valve2048, through fluid path 2049 and down tube 2016. As the fluid exits thetube 2016, it creates a gentle stirring of the cells 2012 such that thefluid around them is mixed with the wash solution, but not so turbulenta motion that a significant number of cells are damaged. The outlet oftube 2016 can, for example, be cut at an angle so that a swirling actionis achieved. The flow rates will depend upon the toughness or fragilityof the cells and the geometry of the vial 2015 b and tubes, and can beadjusted by those skilled in the art, based upon cell viability tests tominimize cell damage and analysis of extracted wastes to ensure thatthere is sufficient agitation and mixing.

Once sufficient wash fluid has been mixed into vial 2015 b, the cellsare allowed to settle. Times on the order of minutes are commonlynecessary, although if the cells are attached to relatively heavysubstrates or magnetic substrates, this time can be significantlyreduced. As mentioned above, centrifuging can increase the separationspeed. It is also possible to employ a filter (not shown) at the tip oftube 2017 or all the way across the area of the vial 2015 b at or belowthe level of the lower end of 2017 so that the cells are constrained tostay in the bottom of the vial 2015 b. (In this case, there would haveto be a penetration, not shown, to allow tube 2106 to reach the bottomof vial 2015 b through the filter.).

As discussed above, after sufficient time, syringe pump 2020 isactivated and the mixed solution, now termed waste is drawn from vial2015 b into syringe 2030 through the one way valve. When syringe 2030 ismoved forward or upward, fluid is driven out the one way valve intowaste bag 2011 or sampling syringe 2033, depending upon position ofstopcock 2041 as discussed above.

A second wash cycle can be performed by repeating the steps of injectionof wash fluid and withdrawal of mixed fluid as discussed above. Eachtime the wash cycle is completed, assuming complete mixing of thecontents of the vial with the wash solution, the transport buffer isdiluted by a specific fraction. If the volume remaining after wastewithdrawal is 1 ml and the volume of wash solution injected is 2 ml,then each wash cycle leaves only 0.33 of the initial transport buffer inthe vial. After 3 cycles, the was buffer has been reduced to 4%, after 6cycles to 0.13%, and after 10 cycles it is down to 15 parts per million.

When the predetermined sequence of washes is completed, for exampleafter a fixed number of washes or when the contents of the samplingsyringe 2033 show that the wash has been sufficient, then the cells areready to be transferred to the delivery syringe 2032. To do this,stopcock 2040 is rotated so that fluid path 2050 is connected to tube2016. As mentioned above, there is no air in the fluid paths, andpreferable syringe 2003 is as empty as possible, meaning that theplunger is all the way up in this diagram or forward in the syringe. Tofill delivery syringe 2032, the plunger is quickly pulled down by pump2022. This sucks cells and fluid out of the bottom of vial 2015 b wherethe cells had settled through tube 2016. To maximize the transfer ofcells, it is best to have the volume fluid of tube 2016, stopcock 2040,and tube 2050 be as low or small as possible in comparison to the volumeof the delivery syringe 2032.

The sequence of wash steps can be controlled by some type of centralcomputer 2029 or sequencer, for instance a laptop computer operatingunder Lab View by National Instruments. The pumps can be independentpumps which are controlled by the central computer is some sort ofdistributed system. Or, the pumps can be part of a single, fullyintegrated system, or the system can have some integration and somedistributed network properties. The stopcocks could be automated andcontrolled by the system. Similarly, the stopcocks and one way valvescould be replaced by pinch valves that are controlled by the system.This could reduce disposable parts costs. It is also possible to replacethe syringe pumps 2021 and 2022 with other pumps, for exampleperistaltic pumps. Peristaltic pumps have the advantage that the one wayvalves can be eliminated. If a dual head, bi-direction peristaltic pumpwith slip clutches in the opposite directions were employed, rotatingthe pump one direction, for example clockwise, would engage the washfluid pump to push fluid into the vial while not moving the waste fluidpump. Then rotating the pump in the counter clockwise direction wouldengage the waste fluid pump to pump out the waste fluid but not engageor rotate the wash fluid pump. Another alternative is to have a singlepump in place of 2020 and 2021 and use an additional stopcock (notshown) or pinch valves to alternatively move wash fluid and waste fluid.This simplification is possible because there is no need to move bothwash and waste fluids at the same time. If a filter is used as mentionedabove, then it can be advantageous to push in wash solution such that itthat overlaps with withdrawal of some waste solution.

A further advantage of the system of FIG. 12 is that it can be operatedfully manually. This is useful in case there is a failure of the pumpingsystem, to save the cost of the pumping system, or for simple procedureswhere the cell washing does not require an automated system. It providesthe benefits of reduced contamination to a manual process that wouldhave otherwise required the use of a sterile hood or glove box.

FIG. 13A illustrates an embodiment of an integrated transport, wash, anddelivery system of the present invention. The outer case 2071 containsthermal management systems such as insulation and ice or active coolingsuch as a Peltier cooling system and a power source (for example, on ormore batteries). The benefit of active cooling is better control oftemperature. The transport case 2071 optionally contains one or moresensors for shock, vibration, temperature, gas or other phenomena thatcould adversely affect the cells, so that if any adverse event occurs,these occurrence(s) are known before the cells are processed or given toa patient.

The wash and delivery subsystem 2070 is sterile on the inside and theoutside, and is contained in a bag (not shown for clarity) to preservethe sterility. In the delivery room, operating room, special procedureslaboratory or wherever the cell delivery is to take place, the wash anddelivery subsystem is removed from the sterile bag and placed in thesterile field.

The wash and delivery subsystem 2070 is positioned so that the syringeis vertical and sufficient time is allowed to for the cells to settle asshown in FIG. 13C. The details of the fluid path are, for example,illustrated in FIG. 13B. The cells are contained in syringe 2034, sealedin by stopcocks 2052 and 2055. There is an optional filter at plunger2035 to prevent cells from setting in fluid path element 2053. Container2010 contains the wash solution and fluid path elements 2050 and 2051are filled with fluid. To perform a wash cycle, stopcock 2052 is turnedto connect syringe 2034 to fluid path 2051. Then stopcock 2055 is turnedto connect fluid path 2053 to either waste container 2011 or samplingsyringe 2033. The syringe pump 2024 pushes down on plunger 2035 andfluid is expelled through one way valve 2054 to the preselected wastecontainer 2011 or sampling syringe 2033. The plunger 2035 moves downuntil sufficient liquid is removed, and stopped before a significantnumber of cells are removed.

Then syringe plunger 2035 is pulled upward, and wash solution is pulledfrom container 2010, through one way valve 2050 and into the neck of thesyringe. This inflowing fluid stream will stir and agitate the cells inthe syringe. An asymmetry of flow at the inlet to the syringe may bedesirable to improve the mixing. This could be as simple as having theneck of the syringe come in off center or at an angle to the vertical toinduce a net rotational force.

Then after sufficient time for the cells to settle, the plunger 2035 ismoved down and the waste mixture is expelled.

This sequence of pulling fluid in from the bottom and expelling it fromthe top can be repeated to wash the cells as many times as desired. Byhaving a sealed wash and delivery subsystem 2070, the fluids in the casewill stay cooler longer, increasing the cell lifetime and reducing thepressure to rush the procedure. Additional thermal mass or a phasechange material can be incorporated to increase the length of time overwhich the cells remain cold.

When the washing is competed, the cells can be delivered without theneed to make any new connections.

To deliver the cells, for example here through a tubing 2059 and aneedle 2060, the wash cycle is stopped, preferably with a littlesolution remaining above the cells, so that all the cells can beeffectively delivered and not trapped in the volume of the syringe neck,tubing 2059, or needle 2060. Then stopcock 2055 is closed and stopcock2052 is adjusted so that syringe 2034 is connected to fluid path 2059.Next, sufficient fluid is delivered to fill fluid path element 2059 andneedle 2060. A sample can be taken by injecting cells out needle 2060 toverify cell viability, type, the lack of contamination, or otherimportant properties or characteristic. This also ensures that the fluidpath is filled with cells.

To deliver the cells, the needle is placed in the tissue and the syringepump, either under direct operator control or as part of a deliverysystem, delivers the selected volume(s) at the selected flow rate(s) atthe selected time(s).

The unit can be place in the sterile field, close the area of use. Theunit can be hung vertically so that cells remain at the neck or outflowof the syringe. As illustrated in FIG. 13D, the needle holder that canbe closed to improve the contamination prevention for the needle. Theneedle is held tip up to minimize any leakage of the cells. However,this may cause some cells to settle away from the tip of the needle. Toavoid this, the needle may be stored horizontally, preferably along thebottom of the subsystem 2070, which is not shown in this figure.

When the procedure is complete, the unit can placed back in its bag,back into outer case and returned to the manufacturer. This procedurecan, for example, enable after-the-fact verification that the cells werenot contaminated during the procedure and allow for reuse andresterilization of the more expensive parts of the system.

As described above, pre-delivery processing of cells and transport ofcell to a delivery system from a processing system (if necessary) ispreferably via a closed or substantially closed system. In that regard,exposure of the cells to a non-sterile environment (for example, byopening a cryovial in non-sterile air) is preferably minimized in boththe number of occurrences and in the length of time. In severalembodiment of the systems of the present invention, the exposure ofcells to a non-sterile environment. Moreover, in case that the cells areexposed to, for example, non-sterile air, the occurrences are limitedto, for example, one or two occurrences and the amount of time of eachsuch occurrence in merely the amount of time required for an operator tomake one or more fluid connections. Further, the systems and devices ofthe present invention can, for example, be used in a cath lab orneurosurgery suite in which it is anticipated that the environmental airwill be quite clean.

While the embodiments of this invention have generally been describedwith respect to the therapeutic delivery or injection of live cells, itwill be apparent to those skilled in the art that this application ofthis invention can provide the benefits described herein to other fluiddelivery situations, for example in the medical field including viraldelivery, large molecule delivery, and drug delivery, and fluid deliveryneeds outside medical treatment arena, including cell, viral, ormolecular delivery in laboratory or industrial applications.

The foregoing description and accompanying drawings set forth thepreferred embodiments of the invention at the present time. Variousmodifications, additions and alternative designs will, of course, becomeapparent to those skilled in the art in light of the foregoing teachingswithout departing from the scope of the invention. The scope of theinvention is indicated by the following claims rather than by theforegoing description. All changes and variations that fall within themeaning and range of equivalency of the claims are to be embraced withintheir scope.

What is claimed is:
 1. A system for processing cells, comprising: acontainer having an inner wall; a plunger adapted to be slidablypositioned within the container, the plunger comprising a filter thatallows fluid to pass therethrough but prevents cells from passingtherethrough; at least one inlet port through which the fluid can enterthe container; at least one outlet port through which the fluid can exitthe container; at least one effluent port through which an effluent canexit the container; a first check valve in fluid connection with theinlet port; and a second check valve in fluid connection with theeffluent port, wherein the inlet port and the effluent port arepositioned on one side of the plunger, the outlet port is positioned onan opposite side of the plunger, and the plunger forms a sealingengagement with the inner wall of the container such that rearwardmotion of the plunger is adapted to draw the fluid into the system viathe inlet port and forward motion of the plunger is adapted to force theeffluent out of the system via the effluent port.
 2. The system of claim1 wherein the effluent port is adapted to effect delivery of the cellstherethrough to a patient.
 3. The system of claim 1 wherein the outletport adapted to effect delivery of the cells therethrough to a patient.4. The system of claim 1 wherein the filter is in fluid connection withthe effluent port to prevent cells from exiting via the effluent port.5. The system of claim 1 wherein the filter separates the cells from theeffluent port.
 6. The system of claim 5 wherein the filter alsoseparates the cells from the inlet port.
 7. The system of claim 6wherein the outlet port is adapted to effect delivery of cellstherethrough to a patient.
 8. The system of claim 7 wherein the outletport is further adapted to be closed during processing of the cellsduring which the fluid enters the system via the inlet port and theeffluent exits the system via the effluent port.
 9. The system of claim8 wherein the effluent port is adapted to be closed when the outlet portis opened to deliver the cells therethrough.
 10. The system of claim 7wherein the inlet port is in fluid connection with a passage through theplunger so that the fluid can enter the plunger and pass through thefilter.
 11. The system of claim 7 wherein the effluent port is in fluidconnection with a passage through the plunger so that the effluent canpass through the filter and exit the effluent port.
 12. The system ofclaim 1 wherein the plunger includes a sealing member adapted to formthe sealing engagement with the interior wall of the container.